U.S. patent application number 17/421365 was filed with the patent office on 2022-03-31 for antibodies and chimeric antigen receptors specific for receptor tyrosine kinase like orphan receptor 1 (ror1).
This patent application is currently assigned to Juno Therapeutics, Inc.. The applicant listed for this patent is Juno Therapeutics, Inc.. Invention is credited to Rupesh AMIN, Jenna BAILEY, Samriti BEDI, Brian BELMONT, Aye CHEN, Andreia COSTA, Stephen Jacob GOLDFLESS, Collin HAUSKINS, Eric JEFFERY, Yue JIANG, Yeonjoo OH, Catherine SIERRA, Madeline WILLIAMS.
Application Number | 20220096651 17/421365 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-31 |
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United States Patent
Application |
20220096651 |
Kind Code |
A1 |
COSTA; Andreia ; et
al. |
March 31, 2022 |
ANTIBODIES AND CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR RECEPTOR
TYROSINE KINASE LIKE ORPHAN RECEPTOR 1 (ROR1)
Abstract
Provided are receptor tyrosine kinase-like orphan receptor 1
(ROR1)-binding molecules, in particular, to human antibodies
specific for ROR1, including antibody fragments. The present
disclosure further relates to recombinant receptors, including
chimeric antigen receptors (CARs) that contain such antibodies or
fragments, and polynucleotides that encode the antibodies,
antigen-binding fragments or receptors specific for ROR1. The
disclosure further relates to genetically engineered cells,
containing such ROR1-binding proteins and receptors, and related
methods and uses thereof in adoptive cell therapy.
Inventors: |
COSTA; Andreia; (Seattle,
WA) ; AMIN; Rupesh; (Seattle, WA) ; BAILEY;
Jenna; (Seattle, WA) ; BEDI; Samriti;
(Newcastle, WA) ; BELMONT; Brian; (Seattle,
WA) ; CHEN; Aye; (Seattle, WA) ; GOLDFLESS;
Stephen Jacob; (Seattle, WA) ; JEFFERY; Eric;
(Seattle, WA) ; JIANG; Yue; (Seattle, WA) ;
OH; Yeonjoo; (Seattle, WA) ; WILLIAMS; Madeline;
(Seattle, WA) ; HAUSKINS; Collin; (Seattle,
WA) ; SIERRA; Catherine; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Juno Therapeutics, Inc. |
Seattle |
WA |
US |
|
|
Assignee: |
Juno Therapeutics, Inc.
Seattle
WA
|
Appl. No.: |
17/421365 |
Filed: |
January 28, 2020 |
PCT Filed: |
January 28, 2020 |
PCT NO: |
PCT/US2020/015489 |
371 Date: |
July 7, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62798456 |
Jan 29, 2019 |
|
|
|
International
Class: |
A61K 47/68 20060101
A61K047/68; A61P 35/00 20060101 A61P035/00; A61K 35/17 20060101
A61K035/17; C12N 5/10 20060101 C12N005/10; C12N 15/63 20060101
C12N015/63; C12N 15/62 20060101 C12N015/62 |
Claims
1. An anti-receptor tyrosine kinase-like orphan receptor 1 (ROR1)
antibody or antigen-binding fragment thereof, comprising: a heavy
chain variable (V.sub.H) region and a light chain variable
(V.sub.L) region, wherein: the V.sub.H region comprises a heavy
chain complementarity determining region 1 (CDR-H1), a heavy chain
complementarity determining region 2 (CDR-H2) and a heavy chain
complementarity determining region 3 (CDR-H3) contained within SEQ
ID NO:112, and the V.sub.L region comprises a light chain
complementarity determining region 1 (CDR-L1), a light chain
complementarity determining region 2 (CDR-L2) and a light chain
complementarity determining region 3 (CDR-L3) contained within SEQ
ID NO:115; the V.sub.H region comprises a CDR-H1, a CDR-H2 and a
CDR-H3 contained within SEQ ID NO:121, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID:
NO 124; the V.sub.H region comprises a CDR-H1, a CDR-H2 and a
CDR-H3 contained within SEQ ID NO:103, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID:
NO 106; or the V.sub.H region comprises a CDR-H1, a CDR-H2 and a
CDR-H3 contained within SEQ ID NO:130, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID:
NO 106.
2. The anti-ROR1 antibody or antigen-binding fragment thereof of
claim 1, wherein the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 contained within SEQ ID NO:112, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID:
NO 115.
3. The anti-ROR1 antibody or antigen-binding fragment thereof of
claim 1, wherein the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 contained within SEQ ID NO:121, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID:
NO 124.
4. The anti-ROR1 antibody or antigen-binding fragment thereof of
claim 1, wherein the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 contained within SEQ ID NO:103, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID:
NO 106.
5. The anti-ROR1 antibody or antigen-binding fragment thereof of
claim 1, wherein the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 contained within SEQ ID NO:130, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID:
NO 106.
6. An anti-ROR1 antibody or antigen-binding fragment thereof,
comprising: a heavy chain variable (V.sub.H) region, and a light
chain variable (V.sub.L) region, wherein the V.sub.H region
comprises a heavy chain complementarity determining region 1
(CDR-H1), a heavy chain complementarity determining region 2
(CDR-H2) and a heavy chain complementarity determining region 3
(CDR-H3) comprising the sequence set forth in SEQ ID NOS:67, 71 and
73, respectively, and the V.sub.L region comprises a light chain
complementarity determining region 1 (CDR-L1), a light chain
complementarity determining region 2 (CDR-L2) and a light chain
complementarity determining region 3 (CDR-L3) comprising the
sequence set forth in SEQ ID NOS:75, 77 and 79, respectively; the
V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising
the sequence set forth in SEQ ID NOS:82, 86 and 88, respectively,
and the V.sub.L region comprises a CDR-L1, a CDR-L2 and a CDR-L3
comprising the sequence set forth in SEQ ID NOS:90, 92 and 94,
respectively; the V.sub.H region comprises a CDR-H1, a CDR-H2 and a
CDR-H3 comprising the sequence set forth in SEQ ID NOS:52, 56 and
58, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:60, 62 and 64, respectively; or the V.sub.H region comprises a
CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in
SEQ ID NOS:52, 97 and 99, respectively, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence
set forth in SEQ ID NOS:60, 62 and 64, respectively.
7. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1, 2 and 6, wherein the V.sub.H region comprises a
CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in
SEQ ID NOS:67, 71 and 73, respectively, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence
set forth in SEQ ID NOS:75, 77 and 79, respectively.
8. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1, 3 and 6, wherein the V.sub.H region comprises a
CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in
SEQ ID NOS:82, 86 and 88, respectively, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence
set forth in SEQ ID NOS:90, 92 and 94, respectively.
9. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1, 4 and 6, wherein the V.sub.H region comprises a
CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in
SEQ ID NOS:52, 56 and 58, respectively, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence
set forth in SEQ ID NOS:60, 62 and 64, respectively.
10. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1, 5 and 6, wherein the V.sub.H region comprises a
CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in
SEQ ID NOS:52, 97 and 99, respectively, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence
set forth in SEQ ID NOS:60, 62 and 64, respectively.
11. The anti-ROR1 antibody or antigen-binding fragment thereof of
claim 1 or claim 6: the V.sub.H region is or comprises an amino
acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO:112, and the V.sub.L region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 115; the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:121, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:124; the V.sub.H
region is or comprises an amino acid sequence having at least at or
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity to SEQ ID NO:103, and the V.sub.L region
is or comprises an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:106; or the V.sub.H region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:130, and the V.sub.L region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:106.
12. An anti-ROR1 antibody or antigen-binding fragment thereof,
comprising: a heavy chain variable (V.sub.H) region and a light
chain variable (V.sub.L) region, wherein: the V.sub.H region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:112, and the V.sub.L region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 115; the V.sub.H region is or comprises
an amino acid sequence having at least at or about 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO:121, and the V.sub.L region is or comprises
an amino acid sequence having at least at or about 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO:124; the V.sub.H region is or comprises an
amino acid sequence having at least at or about 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID NO:103, and the V.sub.L region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:106; or the V.sub.H region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:130, and the V.sub.L region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:106.
13. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1, 2, 6, 7, 11 and 12, wherein the V.sub.H region is
or comprises an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:112, and the V.sub.L region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 115.
14. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1, 3, 6, 8, 11 and 12, wherein the V.sub.H region is
or comprises an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:121, and the V.sub.L region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:124.
15. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1, 4, 6, 9, 11 and 12, wherein the V.sub.H region is
or comprises an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:103, and the V.sub.L region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:106.
16. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1, 5, 6, 10, 11 and 12, wherein the V.sub.H region is
or comprises an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:130, and the V.sub.L region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:106.
17. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1, 6, 11 and 12, wherein: the V.sub.H region and the
V.sub.L region are or comprise the sequence set forth in SEQ ID
NOS: 112 and 115, respectively; the V.sub.H region and the V.sub.L
region are or comprise the sequence set forth in SEQ ID NOS: 121
and 124, respectively; the V.sub.H region and the V.sub.L region
are or comprise the sequence set forth in SEQ ID NOS: 103 and 106,
respectively; or the V.sub.H region and the V.sub.L region are or
comprise the sequence set forth in SEQ ID NOS: 130 and 106,
respectively.
18. An anti-ROR1 antibody or antigen-binding fragment thereof,
comprising: a heavy chain variable (V.sub.H) region and a light
chain variable (V.sub.L) region, wherein: the V.sub.H region and
the V.sub.L region are or comprise the sequence set forth in SEQ ID
NOS: 112 and 115, respectively; the V.sub.H region and the V.sub.L
region are or comprise the sequence set forth in SEQ ID NOS: 121
and 124, respectively; the V.sub.H region and the V.sub.L region
are or comprise the sequence set forth in SEQ ID NOS: 103 and 106,
respectively; or the V.sub.H region and the V.sub.L region are or
comprise the sequence set forth in SEQ ID NOS: 130 and 106,
respectively.
19. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1, 2, 6, 7, 11, 12, 13, 17 and 18, wherein the
V.sub.H region and the V.sub.L region are or comprise the sequence
set forth in SEQ ID NOS: 112 and 115, respectively.
20. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1, 3, 6, 8, 11, 12, 14, 17 and 18, wherein the
V.sub.H region and the V.sub.L region are or comprise the sequence
set forth in SEQ ID NOS:121 and 124, respectively.
21. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1, 4, 6, 9, 11, 12, 15, 17 and 18, wherein the
V.sub.H region and the V.sub.L region are or comprise the sequence
set forth in SEQ ID NOS: 103 and 106, respectively.
22. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1, 5, 6, 10, 11, 12, 16, 17 and 18, wherein the
V.sub.H region and the V.sub.L region are or comprise the sequence
set forth in SEQ ID NOS:130 and 106, respectively.
23. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-22, wherein the antibody is a full length
antibody.
24. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-22, wherein the antibody is an antigen-binding
fragment.
25. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-24, wherein said anti-ROR1 antibody or
antigen-binding fragment thereof is recombinant.
26. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-25, wherein the V.sub.H region and the V.sub.L
region is human or is from a human protein.
27. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-22 and 24-26, wherein the antigen-binding fragment
thereof comprises a single chain Fv (scFv).
28. The anti-ROR1 antibody or antigen-binding fragment thereof of
claim 27, wherein the V.sub.H region is amino-terminal to the
V.sub.L region.
29. The anti-ROR1 antibody or antigen-binding fragment thereof of
claim 27, wherein the V.sub.H region is carboxy-terminal to the
V.sub.L region.
30. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 27-29, when the V.sub.H region and the V.sub.L region
are joined by a flexible linker.
31. The anti-ROR1 antibody or antigen-binding fragment thereof of
claim 30, wherein the flexible linker comprises the sequence set
forth in SEQ ID NO:41.
32. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 27, 28, 30 and 31, wherein the scFv is or comprises
the sequence set forth in SEQ ID NO: 118, 127, 109 or 134, or an
amino acid sequence having at least at or about 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence
identity to SEQ ID NO: 118, 127, 109 or 134.
33. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 27, 28 and 30-32, wherein the scFv is or comprises
the sequence set forth in SEQ ID NO: 118.
34. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 27, 28 and 30-32, wherein the scFv is or comprises
the sequence set forth in SEQ ID NO: 127.
35. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 27, 28 and 30-32, wherein the scFv is or comprises
the sequence set forth in SEQ ID NO: 109.
36. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 27, 28 and 30-32, wherein the scFv is or comprises
the sequence set forth in SEQ ID NO: 134.
37. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-36, wherein said anti-ROR1 antibody or
antigen-binding fragment thereof specifically binds to a human
Receptor tyrosine kinase-like orphan receptor 1 (ROR1) protein.
38. The anti-ROR1 antibody or antigen-binding fragment thereof of
claim 37, wherein the human ROR1 protein comprises an amino acid
sequence set forth in SEQ ID NO: 144, 145 or 146.
39. The anti-ROR1 antibody or antigen-binding fragment thereof of
claim 37 or claim 38, wherein said anti-ROR1 antibody or
antigen-binding fragment thereof specifically binds to an epitope
consisting of the sequence set forth in SEQ ID NO:199 or an epitope
present within the sequence set forth in SEQ ID NO:199.
40. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 37-39, wherein the antibody or antigen-binding
fragment thereof further binds to one or more epitopes consisting
of a sequence selected from among any one of SEQ ID NOS: 200-214 or
an epitope present within a sequence selected from among any one of
SEQ ID NOS: 200-214.
41. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-40, wherein said anti-ROR1 antibody or
antigen-binding fragment thereof does not bind to, is not
cross-reactive to, or binds at a lower level or degree or affinity
to a Receptor tyrosine kinase-like orphan receptor 2 (ROR2)
protein, optionally a human ROR2 protein.
42. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-41, wherein the extent, level or degree or affinity
of binding of said anti-ROR1 antibody or antigen-binding fragment
thereof to a human ROR2 is at least at or about 75%, 80%, 90%, 95%
or 99% less than the extent, level or degree or affinity of binding
to a human ROR1 protein.
43. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-42, wherein the antibody or antigen-binding
fragment thereof binds to human ROR1 protein with an equilibrium
dissociation constant (K.sub.D) of from about 1.times.10.sup.-11 M
to about 1.times.10.sup.-7M.
44. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-43, wherein the antibody or antigen-binding
fragment thereof binds to human ROR1 protein with an equilibrium
dissociation constant (K.sub.D) of from about 1.times.10.sup.-8 M
to about 1.times.10.sup.-7 M.
45. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-43, wherein the antibody or antigen-binding
fragment thereof binds to human ROR1 protein with an equilibrium
dissociation constant (K.sub.D) of from about 5.times.10.sup.-11 M
to about 1.times.10.sup.-10 M.
46. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-45, wherein the antibody or antigen-binding
fragment thereof binds to human ROR1 protein with a dissociation
rate constant (k.sub.d or k.sub.off) of from about
1.times.10.sup.-5 1/s to about 1.times.10.sup.-2 1/s.
47. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-46, wherein the antibody or antigen-binding
fragment thereof binds to human ROR1 protein with a dissociation
rate constant (k.sub.d or k.sub.off) of from about
1.times.10.sup.-3 1/s to about 1.times.10.sup.-2 1/s.
48. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-46, wherein the antibody or antigen-binding
fragment thereof binds to human ROR1 protein with a dissociation
rate constant (k.sub.d or k.sub.off) of from about
1.times.10.sup.-5 1/s to about 1.times.10.sup.-4 1/s.
49. A single chain cell-surface protein, comprising the anti-ROR1
antibody or antigen-binding fragment thereof of any of claims
1-48.
50. A conjugate, comprising the anti-ROR1 antibody or
antigen-binding fragment thereof of any of claims 1-48 and a
heterologous molecule or moiety.
51. The conjugate of claim 50, wherein the heterologous molecule or
moiety is a therapeutic moiety.
52. An anti-ROR1 chimeric antigen receptor (CAR) comprising an
extracellular antigen-binding domain comprising the anti-ROR1
antibody or antigen-binding fragment thereof of any of claims 1-48,
a transmembrane region and an intracellular signaling region.
53. The anti-ROR1 chimeric antigen receptor of claim 52, further
comprising a spacer between the extracellular antigen-binding
domain and the transmembrane domain.
54. The anti-ROR1 chimeric antigen receptor of claim 53, wherein
the spacer comprises at least a portion of an immunoglobulin or a
variant thereof.
55. The anti-ROR1 chimeric antigen receptor of claim 53 or claim
54, wherein the spacer comprises at least a portion of a hinge
region of an immunoglobulin or a variant thereof.
56. The anti-ROR1 chimeric antigen receptor of any of claims 53-55,
wherein the spacer is less than at or about 15 amino acids in
length.
57. The anti-ROR1 chimeric antigen receptor of any of claims 53-56,
wherein the spacer is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1, 26, 27, 29, 31,
32, 33 or 135.
58. The anti-ROR1 chimeric antigen receptor of any of claims 53-57,
wherein the at least a portion of a hinge region comprises all or a
portion of an IgG4 hinge region, optionally a human IgG4 hinge
region, or a variant thereof.
59. The anti-ROR1 chimeric antigen receptor of any of claims 53-57,
wherein the at least a portion of a hinge region comprises all or a
portion of an IgG2 hinge region, optionally a human IgG2 hinge
region, or a variant thereof.
60. The anti-ROR1 chimeric antigen receptor of any of claims 53-58,
wherein the spacer is or comprises the sequence set forth in SEQ ID
NO: 1.
61. The anti-ROR1 chimeric antigen receptor of any of claims 53-58,
wherein the spacer is or comprises the sequence set forth in SEQ ID
NO: 135.
62. The anti-ROR1 chimeric antigen receptor of any of claims 53-55,
wherein the spacer comprises at least a portion of a hinge region
and at least a portion of a CH3 region of an immunoglobulin or a
variant thereof.
63. The anti-ROR1 chimeric antigen receptor of any of claims 53-55
and 62, wherein the at least a portion of a CH3 region comprises
all or a portion of an IgG4 CH3 and/or an IgG2 CH3, wherein the
IgG4 CH3 is optionally a human IgG4 CH3 and the IgG2 CH3 is
optionally a human IgG2 CH3.
64. The anti-ROR1 chimeric antigen receptor of any of claims 53-55,
62 and 63, wherein the spacer is at or about 111, 112, 113, 114,
115, 116, 117, 118, 119, 120, 121, 122, 123, 124 or 125 amino acids
in length, or has a length between any of the foregoing.
65. The anti-ROR1 chimeric antigen receptor of any of claims 53-55
and 62-64, wherein the spacer is at or about 120 amino acids in
length.
66. The anti-ROR1 chimeric antigen receptor of any of claims 53-55
and 62-65, wherein the spacer is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 3 or 138.
67. The anti-ROR1 chimeric antigen receptor of any of claims 53-55
and 62-66, wherein the spacer is or comprises the sequence set
forth in SEQ ID NO: 3 or 138.
68. The anti-ROR1 chimeric antigen receptor of any of claims 53-55,
wherein the spacer comprises at least a portion of a hinge region,
at least a portion of a CH2 and at least a portion of a CH3 region
of an immunoglobulin or a variant thereof.
69. The anti-ROR1 chimeric antigen receptor of any of claims 53-55
and 68, wherein the at least a portion of a CH2 region comprises
all or a portion of an IgG4 CH2 and/or an IgG2 CH2, wherein the
IgG4 CH2 is optionally a human IgG4 CH2 and the IgG2 CH2 is
optionally a human IgG2 CH2.
70. The anti-ROR1 chimeric antigen receptor of any of claims 53-55,
68 and 69, wherein the spacer is at or about 130, 140, 150, 160,
170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227,
228, 229 or 230 amino acids in length, or has a length between any
of the foregoing.
71. The anti-ROR1 chimeric antigen receptor of any of claims 53-55
and 68-70, wherein: one or more of the hinge region, the CH2 region
and the CH3 region comprises all or a portion of a CH2 region and
all or a portion of a CH3 region from human IgG4; or one or more of
the hinge region, the CH2 region and the CH3 region is chimeric and
comprises a hinge, a CH2 region and a CH3 region from human IgG4
and human IgG2; or the spacer comprises a IgG4/2 chimeric hinge
region or a modified IgG4 hinge region comprising at least one
amino acid replacement compared to a human IgG4 hinge; an IgG2/4
chimeric CH2 region; and an IgG4 CH3 region.
72. The anti-ROR1 chimeric antigen receptor of any of claims 53-55
and 68-71, wherein the spacer is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:37 or 194.
73. The anti-ROR1 chimeric antigen receptor of any of claims 53-55
and 68-72, wherein the spacer is or comprises the sequence set
forth in SEQ ID NO: 37 or 194.
74. The anti-ROR1 chimeric antigen receptor of any of claims 52-73,
wherein the transmembrane region is or comprises a transmembrane
domain from CD4, CD28, or CD8.
75. The anti-ROR1 chimeric antigen receptor of any of claims 52-74,
wherein the transmembrane region is or comprises a transmembrane
domain from CD28, optionally a human CD28.
76. The anti-ROR1 chimeric antigen receptor of any of claims 52-75,
wherein the transmembrane domain is or comprises SEQ ID NO: 8 or
149 or an amino acid sequence having at least at or about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ
ID NO: 8 or 149.
77. The anti-ROR1 chimeric antigen receptor of any of claims 52-76,
wherein intracellular signaling region comprises an intracellular
signaling domain capable of inducing a primary activation signal in
a T cell, is a T cell receptor (TCR) component and/or comprises an
immunoreceptor tyrosine-based activation motif (ITAM).
78. The anti-ROR1 chimeric antigen receptor of any of claims 52-77,
wherein the intracellular signaling domain is or comprises a
cytoplasmic signaling domain of a CD3-zeta (CD3.zeta.) chain,
optionally a human CD3.zeta. chain.
79. The anti-ROR1 chimeric antigen receptor of any of claims 52-78,
wherein the intracellular signaling domain is or comprises the
sequence set forth in SEQ ID NO:13, 14 or 15, or an amino acid
sequence having at least at or about 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% sequence identity to SEQ ID NO:13, 14 or
15.
80. The anti-ROR1 chimeric antigen receptor of any of claims 52-79,
wherein the intracellular signaling domain is or comprises the
sequence set forth in SEQ ID NO:13.
81. The anti-ROR1 chimeric antigen receptor of any of claims 52-80,
wherein the intracellular signaling region further comprises a
costimulatory signaling region.
82. The anti-ROR1 chimeric antigen receptor of claim 81, wherein
the costimulatory signaling region is between the transmembrane
region and the intracellular signaling domain.
83. The anti-ROR1 chimeric antigen receptor of claim 81 or claim
82, wherein the costimulatory signaling region comprises an
intracellular signaling domain of a T cell costimulatory molecule
or a signaling portion thereof.
84. The anti-ROR1 chimeric antigen receptor of any of claims 81-83,
wherein the costimulatory signaling region comprises an
intracellular signaling domain of CD28, 4-1BB, or ICOS.
85. The anti-ROR1 chimeric antigen receptor of any of claims 81-84,
wherein the costimulatory signaling region comprises an
intracellular signaling domain of CD28, optionally a human
CD28.
86. The anti-ROR1 chimeric antigen receptor of any of claims 81-85,
wherein the costimulatory signaling region is or comprises the
sequence set forth in SEQ ID NO:10 or an amino acid sequence having
at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% sequence identity to SEQ ID NO:10.
87. The anti-ROR1 chimeric antigen receptor of any of claims 81-84,
wherein the costimulatory signaling region comprises an
intracellular signaling domain of 4-1BB, optionally a human
4-1BB.
88. The anti-ROR1 chimeric antigen receptor of any of claims 81-84
and 87, wherein the costimulatory signaling region is or comprises
the sequence set forth in SEQ ID NO:12 or an amino acid sequence
having at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% sequence identity to SEQ ID NO: 12.
89. The anti-ROR1 chimeric antigen receptor of any of claims 81-88,
wherein the encoded chimeric antigen receptor comprises from its N
to C terminus in order: the extracellular antigen-binding domain,
the spacer, the transmembrane region and the intracellular
signaling region.
90. The anti-ROR1 chimeric antigen receptor of any of claims 81-89,
wherein the antigen-binding domain is an scFv and the encoded
chimeric antigen receptor comprises, from its N to C terminus in
order: an extracellular antigen-binding domain comprising the scFv,
a spacer comprising a modified IgG4 hinge, optionally comprising
the sequence set forth in SEQ ID NO:135; a transmembrane domain,
optionally a transmembrane domain from a human CD28; and an
intracellular signaling region comprising a cytoplasmic signaling
domain of a CD3-zeta (CD3.zeta.) chain and an intracellular
signaling domain of a costimulatory signaling region, optionally
comprising an intracellular signaling domain of 4-1BB.
91. The anti-ROR1 chimeric antigen receptor of any of claims 81-89,
wherein the antigen-binding domain is an scFv and the encoded
chimeric antigen receptor comprises, from its N to C terminus in
order: an extracellular antigen-binding domain comprising the scFv,
a spacer comprising a modified IgG4 hinge-CH3, optionally
comprising the sequence set forth in SEQ ID NO: 138; a
transmembrane domain, optionally a transmembrane domain from a
human CD28; and an intracellular signaling region comprising a
cytoplasmic signaling domain of a CD3-zeta (CD3.zeta.) chain and an
intracellular signaling domain of a costimulatory signaling region,
optionally comprising an intracellular signaling domain of
4-1BB.
92. The anti-ROR1 chimeric antigen receptor of any of claims 52-91,
wherein the anti-ROR1 chimeric antigen receptor is or comprises the
sequence set forth in SEQ ID NO: 184, 185, 186, 187, 188 or 189 or
a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to
the sequence set forth in SEQ ID NO: 184, 185, 186, 187, 188 or
189.
93. The anti-ROR1 chimeric antigen receptor of any of claims 52-92,
wherein the anti-ROR1 chimeric antigen receptor is or comprises the
sequence set forth in SEQ ID NO: 184 or a sequence that exhibits at
least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% sequence identity to the sequence set forth in SEQ
ID NO: 184.
94. The anti-ROR1 chimeric antigen receptor of any of claims 52-93,
wherein the anti-ROR1 chimeric antigen receptor is or comprises the
sequence set forth in SEQ ID NO: 184.
95. The anti-ROR1 chimeric antigen receptor of any of claims 52-92,
wherein the anti-ROR1 chimeric antigen receptor is or comprises the
sequence set forth in SEQ ID NO: 185 or a sequence that exhibits at
least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% sequence identity to the sequence set forth in SEQ
ID NO:185.
96. The anti-ROR1 chimeric antigen receptor of any of claims 52-92
and 95, wherein the anti-ROR1 chimeric antigen receptor is or
comprises the sequence set forth in SEQ ID NO: 185.
97. The anti-ROR1 chimeric antigen receptor of any of claims 52-92,
wherein the anti-ROR1 chimeric antigen receptor is or comprises the
sequence set forth in SEQ ID NO: 186 or a sequence that exhibits at
least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% sequence identity to the sequence set forth in SEQ
ID NO: 186.
98. The anti-ROR1 chimeric antigen receptor of any of claims 52-92
and 97, wherein the anti-ROR1 chimeric antigen receptor is or
comprises the sequence set forth in SEQ ID NO: 186.
99. The anti-ROR1 chimeric antigen receptor of any of claims 52-92,
wherein the anti-ROR1 chimeric antigen receptor is or comprises the
sequence set forth in SEQ ID NO: 187 or a sequence that exhibits at
least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% sequence identity to the sequence set forth in SEQ
ID NO:187.
100. The anti-ROR1 chimeric antigen receptor of any of claims 52-92
and 99, wherein the anti-ROR1 chimeric antigen receptor is or
comprises the sequence set forth in SEQ ID NO: 187.
101. The anti-ROR1 chimeric antigen receptor of any of claims
52-92, wherein the anti-ROR1 chimeric antigen receptor is or
comprises the sequence set forth in SEQ ID NO: 188 or a sequence
that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence
set forth in SEQ ID NO: 188.
102. The anti-ROR1 chimeric antigen receptor of any of claims 52-92
and 101, wherein the anti-ROR1 chimeric antigen receptor is or
comprises the sequence set forth in SEQ ID NO: 188.
103. The anti-ROR1 chimeric antigen receptor of any of claims
52-92, wherein the anti-ROR1 chimeric antigen receptor is or
comprises the sequence set forth in SEQ ID NO: 189 or a sequence
that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence
set forth in SEQ ID NO:189.
104. The anti-ROR1 chimeric antigen receptor of any of claims 52-92
and 103, wherein the anti-ROR1 chimeric antigen receptor is or
comprises the sequence set forth in SEQ ID NO: 189.
105. A polynucleotide comprising a nucleic acid encoding the
anti-ROR1 antibody or antigen-binding domain thereof of any of
claims 1-48.
106. A polynucleotide comprising a nucleic acid encoding the single
chain cell surface protein of claim 49.
107. A polynucleotide comprising a nucleic acid encoding the
conjugate of claim 50 or claim 51.
108. A polynucleotide comprising a nucleic acid encoding the
anti-ROR1 chimeric antigen receptor of any of claims 52-104.
109. The polynucleotide of any of claims 105-108, wherein the
polynucleotide is optimized by splice site elimination.
110. The polynucleotide of any of claims 105-109, wherein the
polynucleotide is codon-optimized for expression in a human
cell.
111. A vector, comprising the polynucleotide of any of claims
105-110.
112. The vector of claim 111, wherein the vector is a viral
vector.
113. The vector of claim 112, wherein the viral vector is a
retroviral vector or a lentiviral vector.
114. A cell comprising the anti-ROR1 antibody or antigen-binding
fragment thereof of any of claims 1-48, the single chain cell
surface protein of claim 49 or the conjugate of claim 50 or claim
51.
115. A cell comprising the anti-ROR1 chimeric antigen receptor of
any of claims 52-104.
116. A cell comprising the polynucleotide of any of claims 105-110,
or the vector of any of 111-113.
117. The cell of any of claims 114-116, that is a lymphocyte.
118. The cell of any of claims 114-116, that is an NK cell or a T
cell.
119. The cell of any of claims 114-118, wherein the cell is a T
cell and the T cell is a CD4+ T cell or a CD8+ T cell.
120. The cell of any of claims 114-119, wherein the cell is a
primary cell obtained from a subject.
121. The cell of any of claims 114-120, wherein, among a plurality
of the cells, less than at or about 10%, at or about 9%, at or
about 8%, at or about 7%, at or about 5%, at or about 4%, at or
about 3%, at or about 2% or at or about 1% of the cells in the
plurality comprise an anti-ROR1 chimeric antigen receptor that
exhibits tonic signaling and/or antigen independent activity or
signaling.
122. A composition comprising the cell of any of claims
114-121.
123. A composition comprising the anti-ROR1 antibody or
antigen-binding fragment thereof of any of claims 1-48, the single
chain cell surface protein of claim 49, the conjugate of claim 50
or claim 51 or the anti-ROR1 chimeric antigen receptor of any of
claims 52-104.
124. The composition of claim 122 or claim 123, further comprising
a pharmaceutically acceptable excipient.
125. The composition of claim 122 or claim 124, wherein the
composition comprises CD4+ and CD8+ T cells and the ratio of CD4+
to CD8+ T cells is from at or about 1:3 to 3:1, optionally at or
about 1:2 to 2:1, optionally at or about 1:1.
126. The composition of any of claims 122, 124 and 125, wherein,
among a plurality of the cells in the composition, less than at or
about 10%, at or about 9%, at or about 8%, at or about 7%, at or
about 5%, at or about 4%, at or about 3%, at or about 2% or at or
about 1% of the cells in the plurality comprise an anti-ROR1
chimeric antigen receptor that exhibits tonic signaling and/or
antigen independent activity or signaling.
127. A method of treatment, comprising administering the cell of
any of claims 114-121 or the composition of any of claims 122-126
to a subject having a disease or disorder associated with ROR1.
128. The cell of any of claims 114-121 or the composition of any of
claims 122-126 for use in treating a disease or disorder associated
with ROR1.
129. Use of the cell of any of claims 114-121 or the composition of
any of claims 122-126 for the manufacture of a medicament for
treating a disease or disorder associated with ROR1.
130. Use of the cell of any of claims 114-121 or the composition of
any of claims 122-126 for the treatment of a disease or disorder
associated with ROR1.
131. A method of treatment, comprising administering the anti-ROR1
antibody or antigen-binding fragment thereof of any of claims 1-48,
the single chain cell surface protein of claim 49, the conjugate of
claim 50 or claim 51, the anti-ROR1 chimeric antigen receptor of
any of claims 52-104, the polynucleotide of any of claims 105-110,
or the vector of any of 111-113 to a subject having a disease or
disorder associated with ROR1.
132. The anti-ROR1 antibody or antigen-binding fragment thereof of
any of claims 1-48, the single chain cell surface protein of claim
49, the conjugate of claim 50 or claim 51, the anti-ROR1 chimeric
antigen receptor of any of claims 52-104, the polynucleotide of any
of claims 105-110, or the vector of any of 111-113 for use in
treating a disease or disorder associated with ROR1.
133. Use of the anti-ROR1 antibody or antigen-binding fragment
thereof of any of claims 1-48, the single chain cell surface
protein of claim 49, the conjugate of claim 50 or claim 51, the
anti-ROR1 chimeric antigen receptor of any of claims 52-104, the
polynucleotide of any of claims 105-110, or the vector of any of
111-113 for the manufacture of a medicament for treating a disease
or disorder associated with ROR1.
134. Use of the anti-ROR1 antibody or antigen-binding fragment
thereof of any of claims 1-48, the single chain cell surface
protein of claim 49, the conjugate of claim 50 or claim 51, the
anti-ROR1 chimeric antigen receptor of any of claims 52-104, the
polynucleotide of any of claims 105-110, or the vector of any of
111-113 for the treatment of a disease or disorder associated with
ROR1.
135. The method, the cell, composition, antibody or antigen-binding
fragment thereof, single chain cell surface protein, conjugate,
chimeric antigen receptor, polynucleotide or vector for use or the
use of any of claims 127-134, wherein the disease or disorder
associated with ROR1 is a cancer.
136. The method, the cell, composition, antibody or antigen-binding
fragment thereof, single chain cell surface protein, conjugate,
chimeric antigen receptor, polynucleotide or vector for use or the
use of claim 135, wherein the cancer is a ROR1-expressing
cancer.
137. The method, the cell, composition, antibody or antigen-binding
fragment thereof, single chain cell surface protein, conjugate,
chimeric antigen receptor, polynucleotide or vector for use or the
use of any of claims 127-136, wherein the cancer is associated with
a ROR1-expressing solid tumor or a ROR1-expressing hematologic
malignancy.
138. The method, the cell, composition, antibody or antigen-binding
fragment thereof, single chain cell surface protein, conjugate,
chimeric antigen receptor, polynucleotide or vector for use or the
use of any of claims 127-137, wherein the cancer is associated with
a ROR1-expressing solid tumor.
139. The method, the cell, composition, antibody or antigen-binding
fragment thereof, single chain cell surface protein, conjugate,
chimeric antigen receptor, polynucleotide or vector for use or the
use of claim 137 or claim 138, wherein the cancer associated with a
solid tumor is selected from the group consisting of neuroblastoma,
renal cell carcinoma, colon cancer, colorectal cancer, breast
cancer, epithelial squamous cell cancer, melanoma, myeloma, stomach
cancer, brain cancer, lung cancer, pancreatic cancer, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, prostate
cancer, testicular cancer, thyroid cancer, uterine cancer, adrenal
cancer and head and neck cancer.
140. The method, the cell, composition, antibody or antigen-binding
fragment thereof, single chain cell surface protein, conjugate,
chimeric antigen receptor, polynucleotide or vector for use or the
use of claim 139, wherein the lung cancer is a non-small cell lung
cancer (NSCLC), lung adenocarcinoma, adenocarcinoma, squamous cell
carcinoma, small cell carcinoma, and atypical carcinoid.
141. The method, the cell, composition, antibody or antigen-binding
fragment thereof, single chain cell surface protein, conjugate,
chimeric antigen receptor, polynucleotide or vector for use or the
use of claim 140, wherein the lung cancer is a NSCLC.
142. The method, the cell, composition, antibody or antigen-binding
fragment thereof, single chain cell surface protein, conjugate,
chimeric antigen receptor, polynucleotide or vector for use or the
use of claim 139, wherein the breast cancer is a triple negative
breast cancer (TNBC).
143. The method, the cell, composition, antibody or antigen-binding
fragment thereof, single chain cell surface protein, conjugate,
chimeric antigen receptor, polynucleotide or vector for use or the
use of any of claims 127-137, wherein the cancer is associated with
a ROR1-expressing hematologic malignancy.
144. The method, the cell, composition, antibody or antigen-binding
fragment thereof, single chain cell surface protein, conjugate,
chimeric antigen receptor, polynucleotide or vector for use or the
use of claim 143, wherein the hematologic malignancy is selected
from the group consisting of B cell leukemia, lymphoma, B cell
chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML),
acute lymphocytic leukemia (ALL), Burkitt's Lymphoma or mantle cell
lymphoma (MCL).
145. A kit comprising the anti-ROR1 antibody or antigen-binding
fragment thereof of any of claims 1-48, the single chain cell
surface protein of claim 49, the conjugate of claim 50 or claim 51,
the anti-ROR1 chimeric antigen receptor of any of claims 52-104,
the polynucleotide of any of claims 105-110, the vector of any of
111-113, the cell of any of claims 114-121 or the composition of
any of claims 122-126, and instructions for use, optionally wherein
the instructions are for administering the anti-ROR1 antibody or
antigen-binding fragment thereof, the single chain cell surface
protein, the conjugate, the anti-ROR1 chimeric antigen receptor,
the cell or the composition, optionally in accord with the method,
the cell, composition, antibody or antigen-binding fragment
thereof, single chain cell surface protein, conjugate, chimeric
antigen receptor, polynucleotide or vector for use or the use of
any of claims 127-144.
146. An article of manufacture comprising the anti-ROR1 antibody or
antigen-binding fragment thereof of any of claims 1-48, the single
chain cell surface protein of claim 49, the conjugate of claim 50
or claim 51, the anti-ROR1 chimeric antigen receptor of any of
claims 52-104, the polynucleotide of any of claims 105-110, the
vector of any of 111-113, the cell of any of claims 114-121 or the
composition of any of claims 122-126 or the kit of claim 145.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. provisional
application No. 62/798,456, filed Jan. 29, 2019, entitled
"ANTIBODIES AND CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR RECEPTOR
TYROSINE KINASE LIKE ORPHAN RECEPTOR 1 (ROR1)," the contents of
which are incorporated by reference in their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled 735042017740SeqList.txt, created Jan. 28, 2020, which
is 225 kilobytes in size. The information in the electronic format
of the Sequence Listing is incorporated by reference in its
entirety.
FIELD
[0003] The present disclosure relates in some aspects to receptor
tyrosine kinase-like orphan receptor 1 (ROR1)-binding molecules, in
particular, to human antibodies specific for ROR1, including
antibody fragments. The present disclosure further relates to
recombinant receptors, including chimeric antigen receptors (CARs)
that contain such antibodies or fragments, and polynucleotides that
encode the antibodies, antigen-binding fragments or receptors
specific for ROR1. The disclosure further relates to genetically
engineered cells, containing such ROR1-binding proteins and
receptors, and related methods and uses thereof in adoptive cell
therapy.
BACKGROUND
[0004] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a
transmembrane receptor expressed during embryogenesis, but
typically not in normal adult cells. ROR1, however, is expressed in
the context of a variety of different cancers, and in some cases,
involved in cell signaling to promote tumor cell survival. Based on
its expression, ROR1 could be a tumor-specific and/or
tumor-associated target for therapy. ROR1-binding molecules,
receptors and cells expressing such molecules are available.
Improved ROR1-binding molecules and engineered ROR1-binding
receptor-expressing cells are needed. Provided are embodiments that
meet such needs.
SUMMARY
[0005] Provided herein is an anti-ROR1 antibody or antigen-binding
fragment thereof, containing: a heavy chain variable (V.sub.H)
region, and a light chain variable (V.sub.L) region, wherein: (i)
the V.sub.H region contains a heavy chain complementarity
determining region 1 (CDR-H1) containing the sequence set forth in
SEQ ID NO: 67, 82 or 52, a heavy chain complementarity determining
region 2 (CDR-H2) containing the sequence set forth in SEQ ID NO:
71, 86, 56 or 97, and a heavy chain complementarity determining
region 3 (CDR-H3) containing the sequence set forth in SEQ ID NO:
73, 88, 58 or 99, and the V.sub.L region contains a light chain
complementarity determining region 1 (CDR-L1) containing the
sequence set forth in SEQ ID NO: 75, 90 or 60, a light chain
complementarity determining region 2 (CDR-L2) containing the
sequence set forth in SEQ ID NO: 77, 92 or 62; and a light chain
complementarity determining region 3 (CDR-L3) containing the
sequence set forth in SEQ ID NO: 79, 94 or 64; or (ii) the V.sub.H
region contains a CDR-H1 containing the sequence set forth in SEQ
ID NO: 65, 80 or 50, a CDR-H2 containing the sequence set forth in
SEQ ID NO: 69, 84, 54 or 95, and a CDR-H3 containing the sequence
set forth in SEQ ID NO: 73, 88, 58 or 99, and the V.sub.L region
contains a CDR-L1 containing the sequence set forth in SEQ ID NO:
75, 90 or 60, a CDR-L2 containing the sequence set forth in SEQ ID
NO: 77, 92 or 62; and a CDR-L3 containing the sequence set forth in
SEQ ID NO: 79, 94 or 64; or (iii) the V.sub.H region contains a
CDR-H1 containing the sequence set forth in SEQ ID NO: 66, 81 or
51, a CDR-H2 containing the sequence set forth in SEQ ID NO: 70,
85, 55 or 96, and a CDR-H3 containing the sequence set forth in SEQ
ID NO: 73, 88, 58 or 99, and the V.sub.L region contains a CDR-L1
containing the sequence set forth in SEQ ID NO: 75, 90 or 60, a
CDR-L2 containing the sequence set forth in SEQ ID NO: 77, 92 or
62; and a CDR-L3 containing the sequence set forth in SEQ ID NO:
79, 94 or 64; or (iv) the V.sub.H region contains a CDR-H1
containing the sequence set forth in SEQ ID NO: 68, 83 or 53, a
CDR-H2 containing the sequence set forth in SEQ ID NO: 72, 87, 57
or 98, and a CDR-H3 containing the sequence set forth in SEQ ID NO:
74, 89, 59 or 100, and the V.sub.L region contains a CDR-L1
containing the sequence set forth in SEQ ID NO: 76, 91 or 61, a
CDR-L2 containing the sequence set forth in SEQ ID NO: 78, 93 or
63; and a CDR-L3 containing the sequence set forth in SEQ ID NO:
79, 94 or 64.
[0006] Also provided herein is an anti-ROR1 antibody or
antigen-binding fragment thereof, containing: a heavy chain
variable (V.sub.H) region, and a light chain variable (V.sub.L)
region, wherein: (i) the V.sub.H region contains a heavy chain
complementarity determining region 1 (CDR-H1), a heavy chain
complementarity determining region 2 (CDR-H2) and a heavy chain
complementarity determining region 3 (CDR-H3) containing the
sequence set forth in SEQ ID NOS:67, 71 and 73, respectively, and
the V.sub.L region contains a light chain complementarity
determining region 1 (CDR-L1), a light chain complementarity
determining region 2 (CDR-L2) and a light chain complementarity
determining region 3 (CDR-L3) containing the sequence set forth in
SEQ ID NOS:75, 77 and 79, respectively; the V.sub.H region contains
a CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence set forth
in SEQ ID NOS:82, 86 and 88, respectively, and the V.sub.L region
contains a CDR-L1, a CDR-L2 and a CDR-L3 containing the sequence
set forth in SEQ ID NOS:90, 92 and 94, respectively; the V.sub.H
region contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the
sequence set forth in SEQ ID NOS:52, 56 and 58, respectively, and
the V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3
containing the sequence set forth in SEQ ID NOS:60, 62 and 64,
respectively; or the V.sub.H region contains a CDR-H1, a CDR-H2 and
a CDR-H3 containing the sequence set forth in SEQ ID NOS:52, 97 and
99, respectively, and the V.sub.L region contains a CDR-L1, a
CDR-L2 and a CDR-L3 containing the sequence set forth in SEQ ID
NOS:60, 62 and 64, respectively; (ii) the V.sub.H region contains a
CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence set forth in
SEQ ID NOS:65, 69 and 73, respectively, and the V.sub.L region
contains a CDR-L1, a CDR-L2 and a CDR-L3 containing the sequence
set forth in SEQ ID NOS:75, 77 and 79, respectively; the V.sub.H
region contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the
sequence set forth in SEQ ID NOS:80, 84 and 88, respectively, and
the V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3
containing the sequence set forth in SEQ ID NOS:90, 92 and 94,
respectively; the V.sub.H region contains a CDR-H1, a CDR-H2 and a
CDR-H3 containing the sequence set forth in SEQ ID NOS:50, 54 and
58, respectively, and the V.sub.L region contains a CDR-L1, a
CDR-L2 and a CDR-L3 containing the sequence set forth in SEQ ID
NOS:60, 62 and 64, respectively; or the V.sub.H region contains a
CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence set forth in
SEQ ID NOS:50, 95 and 99, respectively, and the V.sub.L region
contains a CDR-L1, a CDR-L2 and a CDR-L3 containing the sequence
set forth in SEQ ID NOS:60, 62 and 64, respectively; (iii) the
V.sub.H region contains a CDR-H1, a CDR-H2 and a CDR-H3 containing
the sequence set forth in SEQ ID NOS:66, 70 and 73, respectively,
and the V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3
containing the sequence set forth in SEQ ID NOS:75, 77 and 79,
respectively; the V.sub.H region contains a CDR-H1, a CDR-H2 and a
CDR-H3 containing the sequence set forth in SEQ ID NOS:81, 85 and
88, respectively, and the V.sub.L region contains a CDR-L1, a
CDR-L2 and a CDR-L3 containing the sequence set forth in SEQ ID
NOS:90, 92 and 94, respectively; the V.sub.H region contains a
CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence set forth in
SEQ ID NOS:51, 55 and 58, respectively, and the V.sub.L region
contains a CDR-L1, a CDR-L2 and a CDR-L3 containing the sequence
set forth in SEQ ID NOS:60, 62 and 64, respectively; or the V.sub.H
region contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the
sequence set forth in SEQ ID NOS:51, 96 and 99, respectively, and
the V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3
containing the sequence set forth in SEQ ID NOS:60, 62 and 64,
respectively; (iv) the V.sub.H region contains a CDR-H1, a CDR-H2
and a CDR-H3 containing the sequence set forth in SEQ ID NOS:68, 72
and 74, respectively, and the V.sub.L region contains a CDR-L1, a
CDR-L2 and a CDR-L3 containing the sequence set forth in SEQ ID
NOS:76, 78 and 79, respectively; or the V.sub.H region contains a
CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence set forth in
SEQ ID NOS:83, 87 and 89, respectively, and the V.sub.L region
contains a CDR-L1, a CDR-L2 and a CDR-L3 containing the sequence
set forth in SEQ ID NOS:91, 93 and 94, respectively; the V.sub.H
region contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the
sequence set forth in SEQ ID NOS:53, 57 and 59, respectively, and
the V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3
containing the sequence set forth in SEQ ID NOS:60, 63 and 64,
respectively; or the V.sub.H region contains a CDR-H1, a CDR-H2 and
a CDR-H3 containing the sequence set forth in SEQ ID NOS:53, 98 and
100, respectively, and the V.sub.L region contains a CDR-L1, a
CDR-L2 and a CDR-L3 containing the sequence set forth in SEQ ID
NOS:61, 63 and 64, respectively.
[0007] In some of any such embodiments: (i) the V.sub.H region
contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence
set forth in SEQ ID NOS:67, 71 and 73, respectively, and the
V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3 containing
the sequence set forth in SEQ ID NOS:75, 77 and 79, respectively;
(ii) the V.sub.H region contains a CDR-H1, a CDR-H2 and a CDR-H3
containing the sequence set forth in SEQ ID NOS:65, 69 and 73,
respectively, and the V.sub.L region contains a CDR-L1, a CDR-L2
and a CDR-L3 containing the sequence set forth in SEQ ID NOS:75, 77
and 79, respectively; (iii) the V.sub.H region contains a CDR-H1, a
CDR-H2 and a CDR-H3 containing the sequence set forth in SEQ ID
NOS:66, 70 and 73, respectively, and the V.sub.L region contains a
CDR-L1, a CDR-L2 and a CDR-L3 containing the sequence set forth in
SEQ ID NOS:75, 77 and 79, respectively; or (iv) the V.sub.H region
contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence
set forth in SEQ ID NOS:68, 72 and 74, respectively, and the
V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3 containing
the sequence set forth in SEQ ID NOS:76, 78 and 79,
respectively.
[0008] In some of any such embodiments: (i) the V.sub.H region
contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence
set forth in SEQ ID NOS:82, 86 and 88, respectively, and the
V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3 containing
the sequence set forth in SEQ ID NOS:90, 92 and 94, respectively;
(ii) the V.sub.H region contains a CDR-H1, a CDR-H2 and a CDR-H3
containing the sequence set forth in SEQ ID NOS:80, 84 and 88,
respectively, and the V.sub.L region contains a CDR-L1, a CDR-L2
and a CDR-L3 containing the sequence set forth in SEQ ID NOS:90, 92
and 94, respectively; (iii) the V.sub.H region contains a CDR-H1, a
CDR-H2 and a CDR-H3 containing the sequence set forth in SEQ ID
NOS:81, 85 and 88, respectively, and the V.sub.L region contains a
CDR-L1, a CDR-L2 and a CDR-L3 containing the sequence set forth in
SEQ ID NOS:90, 92 and 94, respectively; or (iv) the V.sub.H region
contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence
set forth in SEQ ID NOS:83, 87 and 89, respectively, and the
V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3 containing
the sequence set forth in SEQ ID NOS:91, 93 and 94,
respectively.
[0009] In some of any embodiments, the V.sub.H region comprises a
CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in
SEQ ID NOS:67, 71 and 73, respectively, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence
set forth in SEQ ID NOS:75, 77 and 79, respectively. In some of any
embodiments, the V.sub.H region comprises a CDR-H1, a CDR-H2 and a
CDR-H3 comprising the sequence set forth in SEQ ID NOS:82, 86 and
88, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:90, 92 and 94, respectively. In some of any such embodiments,
the V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3
comprising the sequence set forth in SEQ ID NOS:52, 56 and 58,
respectively, and the V.sub.L region comprises a CDR-L1, a CDR-L2
and a CDR-L3 comprising the sequence set forth in SEQ ID NOS:60, 62
and 64, respectively. In some of any such embodiments, the V.sub.H
region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the
sequence set forth in SEQ ID NOS:52, 97 and 99, respectively, and
the V.sub.L region comprises a CDR-L1, a CDR-L2 and a CDR-L3
comprising the sequence set forth in SEQ ID NOS:60, 62 and 64,
respectively.
[0010] Also provided herein is an anti-ROR1 antibody or
antigen-binding fragment thereof, containing: a heavy chain
variable (V.sub.H) region and a light chain variable (V.sub.L)
region, wherein: the V.sub.H region contains a heavy chain
complementarity determining region 1 (CDR-H1), a heavy chain
complementarity determining region 2 (CDR-H2) and a heavy chain
complementarity determining region 3 (CDR-H3) contained within SEQ
ID NO: 112, 121, 103 or 130, and the V.sub.L region contains a
light chain complementarity determining region 1 (CDR-L1), a light
chain complementarity determining region 2 (CDR-L2) and a light
chain complementarity determining region 3 (CDR-L3) contained
within SEQ ID NO: 115, 124 or 106.
[0011] Also provided herein is an anti-ROR1 antibody or
antigen-binding fragment thereof, containing: a heavy chain
variable (V.sub.H) region and a light chain variable (V.sub.L)
region, wherein: the V.sub.H region contains a heavy chain
complementarity determining region 1 (CDR-H1), a heavy chain
complementarity determining region 2 (CDR-H2) and a heavy chain
complementarity determining region 3 (CDR-H3) contained within SEQ
ID NO:112, and the V.sub.L region contains a light chain
complementarity determining region 1 (CDR-L1), a light chain
complementarity determining region 2 (CDR-L2) and a light chain
complementarity determining region 3 (CDR-L3) contained within SEQ
ID NO:115; the V.sub.H region contains a CDR-H1, a CDR-H2 and a
CDR-H3 contained within SEQ ID NO:121, and the V.sub.L region
contains a CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID:
NO 124; the V.sub.H region contains a CDR-H1, a CDR-H2 and a CDR-H3
contained within SEQ ID NO:103, and the V.sub.L region contains a
CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID: NO 106; or
the V.sub.H region contains a CDR-H1, a CDR-H2 and a CDR-H3
contained within SEQ ID NO:130, and the V.sub.L region contains a
CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID: NO 106.
[0012] In some of any such embodiments, the V.sub.H region contains
a CDR-H1, a CDR-H2 and a CDR-H3 contained within SEQ ID NO:112, and
the V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3
contained within SEQ ID: NO 115. In some of any such embodiments,
the V.sub.H region contains a CDR-H1, a CDR-H2 and a CDR-H3
contained within SEQ ID NO:121, and the V.sub.L region contains a
CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID: NO 124. In
some of any embodiments, the V.sub.H region comprises a CDR-H1, a
CDR-H2 and a CDR-H3 contained within SEQ ID NO:103, and the V.sub.L
region comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within
SEQ ID: NO 106. In some of any embodiments, the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within SEQ ID
NO:130, and the V.sub.L region comprises a CDR-L1, a CDR-L2 and a
CDR-L3 contained within SEQ ID: NO 106.
[0013] In some of any such embodiments, the V.sub.H region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 112, 121, 103 or 130, and the V.sub.L
region is or contains an amino acid sequence having at least at or
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity to SEQ ID NO: 115, 124 or 106. In some of
any such embodiments, the V.sub.H region is or contains an amino
acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO:112, and the V.sub.L region is or contains an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 115; the V.sub.H region is or contains an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:121, and
the V.sub.L region is or contains an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:124; the V.sub.H
region is or contains an amino acid sequence having at least at or
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity to SEQ ID NO:103, and the V.sub.L region
is or contains an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:106; or the V.sub.H region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:130, and the V.sub.L region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:106.
[0014] In some of any such embodiments, the V.sub.H region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:112, and the V.sub.L region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 115. In some of any such embodiments,
the V.sub.H region is or contains an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:121, and the
V.sub.L region is or contains an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:124. In some of any
embodiments, the V.sub.H region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:103, and the V.sub.L region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:106. In some of any embodiments, the V.sub.H region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:130, and the V.sub.L region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:106.
[0015] Also provided herein is an anti-ROR1 antibody or
antigen-binding fragment thereof, containing: a heavy chain
variable (V.sub.H) region and a light chain variable (V.sub.L)
region, wherein: the V.sub.H region is or contains an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 112, 121, 103 or 130, and the V.sub.L region is or contains an
amino acid sequence having at least at or about 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID NO: 115, 124 or 106.
[0016] Also provided herein is an anti-ROR1 antibody or
antigen-binding fragment thereof, containing: a heavy chain
variable (V.sub.H) region and a light chain variable (V.sub.L)
region, wherein: the V.sub.H region is or contains an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:112, and the V.sub.L region is or contains an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 115; the V.sub.H region is or contains an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:121, and
the V.sub.L region is or contains an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:124; the V.sub.H
region is or contains an amino acid sequence having at least at or
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity to SEQ ID NO:103, and the V.sub.L region
is or contains an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:106; or the V.sub.H region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:130, and the V.sub.L region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:106.
[0017] In some of any such embodiments, the V.sub.H region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:112, and the V.sub.L region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 115. In some of any such embodiments,
the V.sub.H region is or contains an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:121, and the
V.sub.L region is or contains an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:124.
[0018] In some of any such embodiments: the V.sub.H region is or
contains the sequence set forth in SEQ ID NO: 112, 121, 103 or 130,
and the V.sub.L region is or contains the sequence set forth in SEQ
ID NO: 115, 124 or 106. In some of any such embodiments: the
V.sub.H region and the V.sub.L region are or contain the sequence
set forth in SEQ ID NOS: 112 and 115, respectively; the V.sub.H
region and the V.sub.L region are or contain the sequence set forth
in SEQ ID NOS: 121 and 124, respectively; the V.sub.H region and
the V.sub.L region are or contain the sequence set forth in SEQ ID
NOS: 103 and 106, respectively; or the V.sub.H region and the
V.sub.L region are or contain the sequence set forth in SEQ ID NOS:
130 and 106, respectively.
[0019] Also provided herein is an anti-ROR1 antibody or
antigen-binding fragment thereof, containing: a heavy chain
variable (V.sub.H) region and a light chain variable (V.sub.L)
region, wherein: the V.sub.H region is or contains the sequence set
forth in SEQ ID NO: 112, 121, 103 or 130, and the V.sub.L region is
or contains the sequence set forth in SEQ ID NO: 115, 124 or
106.
[0020] Also provided herein is an anti-ROR1 antibody or
antigen-binding fragment thereof, containing: a heavy chain
variable (V.sub.H) region and a light chain variable (V.sub.L)
region, wherein: the V.sub.H region and the V.sub.L region are or
contain the sequence set forth in SEQ ID NOS: 112 and 115,
respectively; the V.sub.H region and the V.sub.L region are or
contain the sequence set forth in SEQ ID NOS: 121 and 124,
respectively; the V.sub.H region and the V.sub.L region are or
contain the sequence set forth in SEQ ID NOS: 103 and 106,
respectively; or the V.sub.H region and the V.sub.L region are or
contain the sequence set forth in SEQ ID NOS: 130 and 106,
respectively.
[0021] In some of any such embodiments, the V.sub.H region and the
V.sub.L region are or contain the sequence set forth in SEQ ID NOS:
112 and 115, respectively. In some of any such embodiments, the
V.sub.H region and the V.sub.L region are or contain the sequence
set forth in SEQ ID NOS:121 and 124, respectively. In some of any
embodiments, the V.sub.H region and the V.sub.L region are or
comprise the sequence set forth in SEQ ID NOS: 103 and 106,
respectively. In some of any embodiments, the V.sub.H region and
the V.sub.L region are or comprise the sequence set forth in SEQ ID
NOS:130 and 106, respectively.
[0022] In some of any such embodiments, the V.sub.H region is or
contains the amino acid sequence encoded by SEQ ID NO: 110, 119,
101 or 128 or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 110,
119, 101 or 128, and the V.sub.L region is or contains the amino
acid sequence encoded by SEQ ID NO: 113, 122 or 104, or a nucleic
acid sequence having at least at or about 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 113, 122 or 104. In some of any
such embodiments, the V.sub.H region is or contains the amino acid
sequence encoded by SEQ ID NO: 111, 120, 102 or 129, and the
V.sub.L region is or contains the amino acid sequence encoded by
SEQ ID NO: 114, 123, 105 or 131. In some of any such embodiments,
the V.sub.H region is or contains the amino acid sequence encoded
by SEQ ID NO: 110 or a nucleic acid sequence having at least at or
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:
110, and the V.sub.L region is or contains the amino acid sequence
encoded by SEQ ID NO: 113, or a nucleic acid sequence having at
least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO: 113. In some of any such embodiments, the V.sub.H region is
or contains the amino acid sequence encoded by SEQ ID NO: 111, and
the V.sub.L region is or contains the amino acid sequence encoded
by SEQ ID NO: 114. In some of any such embodiments, the V.sub.H
region is or contains the amino acid sequence encoded by SEQ ID NO:
119 or a nucleic acid sequence having at least at or about 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 119, and the
V.sub.L region is or contains the amino acid sequence encoded by
SEQ ID NO: 122, or a nucleic acid sequence having at least at or
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:
122. In some of any such embodiments, the V.sub.H region is or
contains the amino acid sequence encoded by SEQ ID NO: 120, and the
V.sub.L region is or contains the amino acid sequence encoded by
SEQ ID NO: 123.
[0023] In some of any embodiments, the antibody is a full length
antibody. In some of any embodiments, the antibody is an
antigen-binding fragment. In some of any such embodiments, said
anti-ROR1 antibody or antigen-binding fragment thereof is isolated.
In some of any such embodiments, said anti-ROR1 antibody or
antigen-binding fragment thereof is recombinant. In some of any
such embodiments, at least a portion of the V.sub.H region and the
V.sub.L region is human or is from a human protein. In some of any
such embodiments, the antigen-binding fragment thereof is or
contains a single chain fragment. In some of any such embodiments,
the antigen-binding fragment thereof is or contains a single chain
Fv (scFv).
[0024] In some of any such embodiments, the V.sub.H region is
amino-terminal to the V.sub.L region. In some of any such
embodiments, the V.sub.H region is carboxy-terminal to the V.sub.L
region. In some of any such embodiments, the V.sub.H region and the
V.sub.L region are joined by a flexible linker. In some of any such
embodiments, the flexible linker contains the sequence set forth in
SEQ ID NO:41.
[0025] In some of any such embodiments, the scFv is or contains the
sequence set forth in SEQ ID NO: 118, 127, 109 or 134, or an amino
acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence
identity to SEQ ID NO: 118, 127, 109 or 134. In some of any such
embodiments, the scFv is or contains the sequence set forth in SEQ
ID NO: 118. In some of any such embodiments, the scFv is or
contains the sequence set forth in SEQ ID NO: 127. In some of any
embodiments, the scFv is or comprises the sequence set forth in SEQ
ID NO: 109. In some of any embodiments, the scFv is or comprises
the sequence set forth in SEQ ID NO: 134.
[0026] In some of any such embodiments, the scFv is or contains the
amino acid sequence encoded by SEQ ID NO: 116, 125, 107 or 132 or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 116, 125, 107 or 132.
In some of any such embodiments, the scFv is or contains the amino
acid sequence encoded by SEQ ID NO: 117, 126, 108 or 133. In some
of any such embodiments, the scFv is or contains the amino acid
sequence encoded by SEQ ID NO: 116 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 116. In some of any such embodiments, the
scFv is or contains the amino acid sequence encoded by SEQ ID NO:
117. In some of any such embodiments, the scFv is or contains the
amino acid sequence encoded by SEQ ID NO: 125 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 125. In some of any such embodiments,
the scFv is or contains the amino acid sequence encoded by SEQ ID
NO: 126.
[0027] In some of any such embodiments, the anti-ROR1 antibody or
fragment further contains at least a portion of an immunoglobulin
constant region or a variant thereof. In some of any such
embodiments, the portion of an immunoglobulin constant region
contains at least a portion of a hinge region or a variant thereof.
In some of any such embodiments, the at least a portion of an
immunoglobulin constant region or a variant thereof contains at
least a portion of a C.sub.H2 region or a variant thereof. In some
of any such embodiments, the at least a portion of an
immunoglobulin constant region or a variant thereof contains at
least a portion of a C.sub.H3 region or a variant thereof. In some
of any such embodiments, the at least a portion of an
immunoglobulin constant region or a variant thereof contains at
least a portion of a C.sub.H2 region and/or a C.sub.H3 region or a
variant thereof. In some of any such embodiments, the at least a
portion of an immunoglobulin constant region or a variant thereof
is human or from a human protein or a variant thereof.
[0028] In some of any such embodiments, said anti-ROR1 antibody or
antigen-binding fragment thereof specifically binds to a Receptor
tyrosine kinase-like orphan receptor 1 (ROR1) protein. In some of
any such embodiments, said anti-ROR1 antibody or antigen-binding
fragment thereof specifically binds to a human ROR1 protein. In
some of any such embodiments, the human ROR1 protein contains an
amino acid sequence set forth in SEQ ID NO: 144, 145 or 146.
[0029] In some of any embodiments, the anti-ROR1 antibody or
antigen-binding fragment thereof specifically binds to an epitope
consisting of the sequence set forth in SEQ ID NO:199 or an epitope
present within the sequence set forth in SEQ ID NO:199. In some of
any embodiments, the antibody or antigen-binding fragment thereof
further binds to one or more epitopes consisting of a sequence
selected from among any one of SEQ ID NOS: 200-214 or an epitope
present within a sequence selected from among any one of SEQ ID
NOS: 200-214. In some of any embodiments, the one or more epitopes
comprises a conformational epitope.
[0030] In some of any such embodiments, said anti-ROR1 antibody or
antigen-binding fragment thereof does not bind to, is not
cross-reactive to, or binds at a lower level or degree or affinity
to a Receptor tyrosine kinase-like orphan receptor 2 (ROR2)
protein. In some of any such embodiments, said anti-ROR1 antibody
or antigen-binding fragment thereof does not bind to, is not
cross-reactive to, or binds at a lower level or degree or affinity
to a human ROR2 protein. In some of any such embodiments, the
extent, level or degree or affinity of binding of said anti-ROR1
antibody or antigen-binding fragment thereof to a human ROR2 is at
least at or about 75%, 80%, 90%, 95% or 99% less than the extent,
level or degree or affinity of binding to a human ROR1 protein.
[0031] In some of any embodiments, the antibody or antigen-binding
fragment thereof binds to human ROR1 protein with an equilibrium
dissociation constant (K.sub.D) of from about 1.times.10.sup.-11 M
to about 1.times.10.sup.-7 M. In some of any embodiments, the
antibody or antigen-binding fragment thereof binds to human ROR1
protein with an equilibrium dissociation constant (K.sub.D) of from
about 1.times.10.sup.-8 M to about 1.times.10.sup.-7 M. In some of
any embodiments, the antibody or antigen-binding fragment thereof
binds to human ROR1 protein with an equilibrium dissociation
constant (K.sub.D) of from about 5.times.10.sup.-11 M to about
1.times.10.sup.10 M.
[0032] In some of any embodiments, the antibody or antigen-binding
fragment thereof binds to human ROR1 protein with an dissociation
rate constant (k.sub.d or k.sub.off) of from about
1.times.10.sup.-5 1/s to about 1.times.10.sup.-2 1/s. In some of
any embodiments, the antibody or antigen-binding fragment thereof
binds to human ROR1 protein with an dissociation rate constant
(k.sub.d or k.sub.off) of from about 1.times.10.sup.-3 1/s to about
1.times.10.sup.-2 1/s. In some of any embodiments, the antibody or
antigen-binding fragment thereof binds to human ROR1 protein with
an dissociation rate constant (k.sub.d or k.sub.off) of from about
1.times.10.sup.-5 1/s to about 1.times.10.sup.-4 1/s.
[0033] Also provided herein is a single chain cell-surface protein,
containing the anti-ROR1 antibody or antigen-binding fragment
thereof of any of the embodiments provided herein.
[0034] Also provided herein is a conjugate, containing the
anti-ROR1 antibody or antigen-binding fragment thereof of any of
the embodiments provided herein and a heterologous molecule or
moiety. In some of any such embodiments, the heterologous molecule
or moiety is a therapeutic moiety.
[0035] Also provided herein is an anti-ROR1 chimeric antigen
receptor (CAR) containing an extracellular antigen-binding domain
containing the anti-ROR1 antibody or antigen-binding fragment
thereof of any of the embodiments provided herein and an
intracellular signaling region.
[0036] Also provided herein is an anti-ROR1 chimeric antigen
receptor (CAR) containing an extracellular antigen-binding domain
containing the anti-ROR1 antibody or antigen-binding fragment
thereof of any of the embodiments provided herein, a transmembrane
region and an intracellular signaling region.
[0037] Also provided herein is an anti-ROR1 chimeric antigen
receptor (CAR) containing: an extracellular antigen-binding domain
containing a heavy chain variable (V.sub.H) region, and a light
chain variable (V.sub.L) region, and an intracellular signaling
region, wherein: (i) the V.sub.H region contains a heavy chain
complementarity determining region 1 (CDR-H1) containing the
sequence set forth in SEQ ID NO: 67, 82 or 52, a heavy chain
complementarity determining region 2 (CDR-H2) containing the
sequence set forth in SEQ ID NO: 71, 86, 56 or 97, and a heavy
chain complementarity determining region 3 (CDR-H3) containing the
sequence set forth in SEQ ID NO: 73, 88, 58 or 99, and the V.sub.L
region contains a light chain complementarity determining region 1
(CDR-L1) containing the sequence set forth in SEQ ID NO: 75, 90 or
60, a light chain complementarity determining region 2 (CDR-L2)
containing the sequence set forth in SEQ ID NO: 77, 92 or 62; and a
light chain complementarity determining region 3 (CDR-L3)
containing the sequence set forth in SEQ ID NO: 79, 94 or 64; or
(ii) the V.sub.H region contains a CDR-H1 containing the sequence
set forth in SEQ ID NO: 65, 80 or 50, a CDR-H2 containing the
sequence set forth in SEQ ID NO: 69, 84, 54 or 95, and a CDR-H3
containing the sequence set forth in SEQ ID NO: 73, 88, 58 or 99,
and the V.sub.L region contains a CDR-L1 containing the sequence
set forth in SEQ ID NO: 75, 90 or 60, a CDR-L2 containing the
sequence set forth in SEQ ID NO: 77, 92 or 62; and a CDR-L3
containing the sequence set forth in SEQ ID NO: 79, 94 or 64; or
(iii) the V.sub.H region contains a CDR-H1 containing the sequence
set forth in SEQ ID NO: 66, 81 or 51, a CDR-H2 containing the
sequence set forth in SEQ ID NO: 70, 85, 55 or 96, and a CDR-H3
containing the sequence set forth in SEQ ID NO: 73, 88, 58 or 99,
and the V.sub.L region contains a CDR-L1 containing the sequence
set forth in SEQ ID NO: 75, 90 or 60, a CDR-L2 containing the
sequence set forth in SEQ ID NO: 77, 92 or 62; and a CDR-L3
containing the sequence set forth in SEQ ID NO: 79, 94 or 64; or
(iv) the V.sub.H region contains a CDR-H1 containing the sequence
set forth in SEQ ID NO: 68, 83 or 53, a CDR-H2 containing the
sequence set forth in SEQ ID NO: 72, 87, 57 or 98, and a CDR-H3
containing the sequence set forth in SEQ ID NO: 74, 89, 59 or 100,
and the V.sub.L region contains a CDR-L1 containing the sequence
set forth in SEQ ID NO: 76, 91 or 61, a CDR-L2 containing the
sequence set forth in SEQ ID NO: 78, 93 or 63; and a CDR-L3
containing the sequence set forth in SEQ ID NO: 79, 94 or 64.
[0038] Also provided herein is an anti-ROR1 chimeric antigen
receptor (CAR) containing: an extracellular antigen-binding domain
containing a heavy chain variable (V.sub.H) region, and a light
chain variable (V.sub.L) region, and an intracellular signaling
region, wherein: (i) the V.sub.H region contains a heavy chain
complementarity determining region 1 (CDR-H1), a heavy chain
complementarity determining region 2 (CDR-H2) and a heavy chain
complementarity determining region 3 (CDR-H3) containing the
sequence set forth in SEQ ID NOS:67, 71 and 73, respectively, and
the V.sub.L region contains a light chain complementarity
determining region 1 (CDR-L1), a light chain complementarity
determining region 2 (CDR-L2) and a light chain complementarity
determining region 3 (CDR-L3) containing the sequence set forth in
SEQ ID NOS:75, 77 and 79, respectively; the V.sub.H region contains
a CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence set forth
in SEQ ID NOS:82, 86 and 88, respectively, and the V.sub.L region
contains a CDR-L1, a CDR-L2 and a CDR-L3 containing the sequence
set forth in SEQ ID NOS:90, 92 and 94, respectively; the V.sub.H
region contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the
sequence set forth in SEQ ID NOS:52, 56 and 58, respectively, and
the V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3
containing the sequence set forth in SEQ ID NOS:60, 62 and 64,
respectively; or the V.sub.H region contains a CDR-H1, a CDR-H2 and
a CDR-H3 containing the sequence set forth in SEQ ID NOS:52, 97 and
99, respectively, and the V.sub.L region contains a CDR-L1, a
CDR-L2 and a CDR-L3 containing the sequence set forth in SEQ ID
NOS:60, 62 and 64, respectively; (ii) the V.sub.H region contains a
CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence set forth in
SEQ ID NOS:65, 69 and 73, respectively, and the V.sub.L region
contains a CDR-L1, a CDR-L2 and a CDR-L3 containing the sequence
set forth in SEQ ID NOS:75, 77 and 79, respectively; the V.sub.H
region contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the
sequence set forth in SEQ ID NOS:80, 84 and 88, respectively, and
the V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3
containing the sequence set forth in SEQ ID NOS:90, 92 and 94,
respectively; the V.sub.H region contains a CDR-H1, a CDR-H2 and a
CDR-H3 containing the sequence set forth in SEQ ID NOS:50, 54 and
58, respectively, and the V.sub.L region contains a CDR-L1, a
CDR-L2 and a CDR-L3 containing the sequence set forth in SEQ ID
NOS:60, 62 and 64, respectively; or the V.sub.H region contains a
CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence set forth in
SEQ ID NOS:50, 95 and 99, respectively, and the V.sub.L region
contains a CDR-L1, a CDR-L2 and a CDR-L3 containing the sequence
set forth in SEQ ID NOS:60, 62 and 64, respectively; (iii) the
V.sub.H region contains a CDR-H1, a CDR-H2 and a CDR-H3 containing
the sequence set forth in SEQ ID NOS:66, 70 and 73, respectively,
and the V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3
containing the sequence set forth in SEQ ID NOS:75, 77 and 79,
respectively; the V.sub.H region contains a CDR-H1, a CDR-H2 and a
CDR-H3 containing the sequence set forth in SEQ ID NOS:81, 85 and
88, respectively, and the V.sub.L region contains a CDR-L1, a
CDR-L2 and a CDR-L3 containing the sequence set forth in SEQ ID
NOS:90, 92 and 94, respectively; the V.sub.H region contains a
CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence set forth in
SEQ ID NOS:51, 55 and 58, respectively, and the V.sub.L region
contains a CDR-L1, a CDR-L2 and a CDR-L3 containing the sequence
set forth in SEQ ID NOS:60, 62 and 64, respectively; or the V.sub.H
region contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the
sequence set forth in SEQ ID NOS:51, 96 and 99, respectively, and
the V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3
containing the sequence set forth in SEQ ID NOS:60, 62 and 64,
respectively; (iv) the V.sub.H region contains a CDR-H1, a CDR-H2
and a CDR-H3 containing the sequence set forth in SEQ ID NOS:68, 72
and 74, respectively, and the V.sub.L region contains a CDR-L1, a
CDR-L2 and a CDR-L3 containing the sequence set forth in SEQ ID
NOS:76, 78 and 79, respectively; or the V.sub.H region contains a
CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence set forth in
SEQ ID NOS:83, 87 and 89, respectively, and the V.sub.L region
contains a CDR-L1, a CDR-L2 and a CDR-L3 containing the sequence
set forth in SEQ ID NOS:91, 93 and 94, respectively; the V.sub.H
region contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the
sequence set forth in SEQ ID NOS:53, 57 and 59, respectively, and
the V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3
containing the sequence set forth in SEQ ID NOS:60, 63 and 64,
respectively; or the V.sub.H region contains a CDR-H1, a CDR-H2 and
a CDR-H3 containing the sequence set forth in SEQ ID NOS:53, 98 and
100, respectively, and the V.sub.L region contains a CDR-L1, a
CDR-L2 and a CDR-L3 containing the sequence set forth in SEQ ID
NOS:61, 63 and 64, respectively.
[0039] In some of any such embodiments: (i) the V.sub.H region
contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence
set forth in SEQ ID NOS:67, 71 and 73, respectively, and the
V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3 containing
the sequence set forth in SEQ ID NOS:75, 77 and 79, respectively;
(ii) the V.sub.H region contains a CDR-H1, a CDR-H2 and a CDR-H3
containing the sequence set forth in SEQ ID NOS:65, 69 and 73,
respectively, and the V.sub.L region contains a CDR-L1, a CDR-L2
and a CDR-L3 containing the sequence set forth in SEQ ID NOS:75, 77
and 79, respectively; (iii) the V.sub.H region contains a CDR-H1, a
CDR-H2 and a CDR-H3 containing the sequence set forth in SEQ ID
NOS:66, 70 and 73, respectively, and the V.sub.L region contains a
CDR-L1, a CDR-L2 and a CDR-L3 containing the sequence set forth in
SEQ ID NOS:75, 77 and 79, respectively; or (iv) the V.sub.H region
contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence
set forth in SEQ ID NOS:68, 72 and 74, respectively, and the
V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3 containing
the sequence set forth in SEQ ID NOS:76, 78 and 79,
respectively.
[0040] In some of any such embodiments: (i) the V.sub.H region
contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence
set forth in SEQ ID NOS:82, 86 and 88, respectively, and the
V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3 containing
the sequence set forth in SEQ ID NOS:90, 92 and 94, respectively;
(ii) the V.sub.H region contains a CDR-H1, a CDR-H2 and a CDR-H3
containing the sequence set forth in SEQ ID NOS:80, 84 and 88,
respectively, and the V.sub.L region contains a CDR-L1, a CDR-L2
and a CDR-L3 containing the sequence set forth in SEQ ID NOS:90, 92
and 94, respectively; (iii) the V.sub.H region contains a CDR-H1, a
CDR-H2 and a CDR-H3 containing the sequence set forth in SEQ ID
NOS:81, 85 and 88, respectively, and the V.sub.L region contains a
CDR-L1, a CDR-L2 and a CDR-L3 containing the sequence set forth in
SEQ ID NOS:90, 92 and 94, respectively; or (iv) the V.sub.H region
contains a CDR-H1, a CDR-H2 and a CDR-H3 containing the sequence
set forth in SEQ ID NOS:83, 87 and 89, respectively, and the
V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3 containing
the sequence set forth in SEQ ID NOS:91, 93 and 94,
respectively.
[0041] Also provided herein is an anti-ROR1 chimeric antigen
receptor (CAR) containing: an extracellular antigen-binding domain
containing a heavy chain variable (V.sub.H) region and a light
chain variable (V.sub.L) region, and an intracellular signaling
region, wherein: the V.sub.H region contains a heavy chain
complementarity determining region 1 (CDR-H1), a heavy chain
complementarity determining region 2 (CDR-H2) and a heavy chain
complementarity determining region 3 (CDR-H3) contained within SEQ
ID NO: 112, 121, 103 or 130, and the V.sub.L region contains a
light chain complementarity determining region 1 (CDR-L1), a light
chain complementarity determining region 2 (CDR-L2) and a light
chain complementarity determining region 3 (CDR-L3) contained
within SEQ ID NO: 115, 124 or 106.
[0042] Also provided herein is an anti-ROR1 chimeric antigen
receptor (CAR) containing: an extracellular antigen-binding domain
containing a heavy chain variable (V.sub.H) region and a light
chain variable (V.sub.L) region, and an intracellular signaling
region, wherein: the V.sub.H region contains a heavy chain
complementarity determining region 1 (CDR-H1), a heavy chain
complementarity determining region 2 (CDR-H2) and a heavy chain
complementarity determining region 3 (CDR-H3) contained within SEQ
ID NO:112, and the V.sub.L region contains a light chain
complementarity determining region 1 (CDR-L1), a light chain
complementarity determining region 2 (CDR-L2) and a light chain
complementarity determining region 3 (CDR-L3) contained within SEQ
ID NO:115; the V.sub.H region contains a CDR-H1, a CDR-H2 and a
CDR-H3 contained within SEQ ID NO:121, and the V.sub.L region
contains a CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID:
NO 124; the V.sub.H region contains a CDR-H1, a CDR-H2 and a CDR-H3
contained within SEQ ID NO:103, and the V.sub.L region contains a
CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID: NO 106; or
the V.sub.H region contains a CDR-H1, a CDR-H2 and a CDR-H3
contained within SEQ ID NO:130, and the V.sub.L region contains a
CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID: NO 106.
[0043] In some of any such embodiments, the V.sub.H region contains
a CDR-H1, a CDR-H2 and a CDR-H3 contained within SEQ ID NO:112, and
the V.sub.L region contains a CDR-L1, a CDR-L2 and a CDR-L3
contained within SEQ ID: NO 115. In some of any such embodiments,
the V.sub.H region contains a CDR-H1, a CDR-H2 and a CDR-H3
contained within SEQ ID NO:121, and the V.sub.L region contains a
CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID: NO 124.
[0044] In some of any such embodiments: the V.sub.H region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 112, 121, 103 or 130, and the V.sub.L
region is or contains an amino acid sequence having at least at or
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity to SEQ ID NO: 115, 124 or 106. In some of
any such embodiments: the V.sub.H region is or contains an amino
acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO:112, and the V.sub.L region is or contains an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 115; the V.sub.H region is or contains an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:121, and
the V.sub.L region is or contains an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:124; the V.sub.H
region is or contains an amino acid sequence having at least at or
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity to SEQ ID NO:103, and the V.sub.L region
is or contains an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:106; or the V.sub.H region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:130, and the V.sub.L region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:106.
[0045] In some of any such embodiments, the V.sub.H region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:112, and the V.sub.L region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 115. In some of any such embodiments,
the V.sub.H region is or contains an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:121, and the
V.sub.L region is or contains an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:124.
[0046] Also provided herein is an anti-ROR1 chimeric antigen
receptor (CAR) containing: an extracellular antigen-binding domain
containing a heavy chain variable (V.sub.H) region and a light
chain variable (V.sub.L) region, and an intracellular signaling
region, wherein: the V.sub.H region is or contains an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 112, 121, 103 or 130, and the V.sub.L region is or contains an
amino acid sequence having at least at or about 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID NO: 115, 124 or 106.
[0047] Also provided herein is an anti-ROR1 chimeric antigen
receptor (CAR) containing: an extracellular antigen-binding domain
containing a heavy chain variable (V.sub.H) region and a light
chain variable (V.sub.L) region, and an intracellular signaling
region, wherein: the V.sub.H region is or contains an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:112, and the V.sub.L region is or contains an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 115; the V.sub.H region is or contains an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:121, and
the V.sub.L region is or contains an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:124; the V.sub.H
region is or contains an amino acid sequence having at least at or
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity to SEQ ID NO:103, and the V.sub.L region
is or contains an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:106; or the V.sub.H region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:130, and the V.sub.L region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:106.
[0048] In some of any such embodiments, the V.sub.H region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:112, and the V.sub.L region is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 115. In some of any such embodiments,
the V.sub.H region is or contains an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:121, and the
V.sub.L region is or contains an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:124.
[0049] In some of any such embodiments: the V.sub.H region is or
contains the sequence set forth in SEQ ID NO: 112, 121, 103 or 130,
and the V.sub.L region is or contains the sequence set forth in SEQ
ID NO: 115, 124 or 106.
[0050] In some of any such embodiments: the V.sub.H region and the
V.sub.L region are or contain the sequence set forth in SEQ ID NOS:
112 and 115, respectively; the V.sub.H region and the V.sub.L
region are or contain the sequence set forth in SEQ ID NOS: 121 and
124, respectively; the V.sub.H region and the V.sub.L region are or
contain the sequence set forth in SEQ ID NOS: 103 and 106,
respectively; or the V.sub.H region and the V.sub.L region are or
contain the sequence set forth in SEQ ID NOS: 130 and 106,
respectively.
[0051] Also provided herein is an anti-ROR1 chimeric antigen
receptor (CAR) containing: an extracellular antigen-binding domain
containing a heavy chain variable (V.sub.H) region and a light
chain variable (V.sub.L) region, and an intracellular signaling
region, wherein: the V.sub.H region is or contains the sequence set
forth in SEQ ID NO: 112, 121, 103 or 130, and the V.sub.L region is
or contains the sequence set forth in SEQ ID NO: 115, 124 or
106.
[0052] Also provided herein is an anti-ROR1 chimeric antigen
receptor (CAR) containing: an extracellular antigen-binding domain
containing a heavy chain variable (V.sub.H) region and a light
chain variable (V.sub.L) region, and an intracellular signaling
region, wherein: the V.sub.H region and the V.sub.L region are or
contain the sequence set forth in SEQ ID NOS: 112 and 115,
respectively; the V.sub.H region and the V.sub.L region are or
contain the sequence set forth in SEQ ID NOS: 121 and 124,
respectively; the V.sub.H region and the V.sub.L region are or
contain the sequence set forth in SEQ ID NOS: 103 and 106,
respectively; or the V.sub.H region and the V.sub.L region are or
contain the sequence set forth in SEQ ID NOS: 130 and 106,
respectively.
[0053] In some of any such embodiments, the V.sub.H region and the
V.sub.L region are or contain the sequence set forth in SEQ ID NOS:
112 and 115, respectively. In some of any such embodiments, the
V.sub.H region and the V.sub.L region are or contain the sequence
set forth in SEQ ID NOS:121 and 124, respectively.
[0054] In some of any such embodiments, the V.sub.H region is or
contains the amino acid sequence encoded by SEQ ID NO: 110, 119,
101 or 128 or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 110,
119, 101 or 128, and the V.sub.L region is or contains the amino
acid sequence encoded by SEQ ID NO: 113, 122 or 104, or a nucleic
acid sequence having at least at or about 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 113, 122 or 104. In some of any
such embodiments, the V.sub.H region is or contains the amino acid
sequence encoded by SEQ ID NO: 111, 120, 102 or 129, and the
V.sub.L region is or contains the amino acid sequence encoded by
SEQ ID NO: 114, 123, 105 or 131. In some of any such embodiments,
the V.sub.H region is or contains the amino acid sequence encoded
by SEQ ID NO: 110 or a nucleic acid sequence having at least at or
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:
110, and the V.sub.L region is or contains the amino acid sequence
encoded by SEQ ID NO: 113, or a nucleic acid sequence having at
least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO: 113. In some of any such embodiments, the V.sub.H region is
or contains the amino acid sequence encoded by SEQ ID NO: 111, and
the V.sub.L region is or contains the amino acid sequence encoded
by SEQ ID NO: 114. In some of any such embodiments, the V.sub.H
region is or contains the amino acid sequence encoded by SEQ ID NO:
119 or a nucleic acid sequence having at least at or about 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 119, and the
V.sub.L region is or contains the amino acid sequence encoded by
SEQ ID NO: 122, or a nucleic acid sequence having at least at or
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:
122. In some of any such embodiments, the V.sub.H region is or
contains the amino acid sequence encoded by SEQ ID NO: 120, and the
V.sub.L region is or contains the amino acid sequence encoded by
SEQ ID NO: 123.
[0055] In some of any such embodiments, said anti-ROR1 antibody or
antigen-binding fragment thereof is isolated. In some of any such
embodiments, said anti-ROR1 antibody or antigen-binding fragment
thereof is recombinant. In some of any such embodiments, at least a
portion of the V.sub.H region and the V.sub.L region is human or is
from a human protein.
[0056] In some of any such embodiments, the antigen-binding
fragment thereof is or contains a single chain fragment. In some of
any such embodiments, the antigen-binding fragment thereof is or
contains a single chain Fv (scFv).
[0057] In some of any such embodiments, the V.sub.H region is
amino-terminal to the V.sub.L region. In some of any such
embodiments, the V.sub.H region is carboxy-terminal to the V.sub.L
region. In some of any such embodiments, the V.sub.H region and the
V.sub.L region are joined by a flexible linker. In some of any such
embodiments, the flexible linker contains the sequence set forth in
SEQ ID NO:41.
[0058] In some of any such embodiments, the scFv is or contains the
sequence set forth in SEQ ID NO: 118, 127, 109 or 134, or an amino
acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence
identity to SEQ ID NO: 118, 127, 109 or 134. In some of any such
embodiments, the scFv is or contains the sequence set forth in SEQ
ID NO: 118. In some of any such embodiments, the scFv is or
contains the sequence set forth in SEQ ID NO: 127. In some of any
such embodiments, the scFv is or contains the amino acid sequence
encoded by SEQ ID NO: 116, 125, 107 or 132 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 116, 125, 107 or 132. In some of any
such embodiments, the scFv is or contains the amino acid sequence
encoded by SEQ ID NO: 117, 126, 108 or 133. In some of any such
embodiments, the scFv is or contains the amino acid sequence
encoded by SEQ ID NO: 116 or a nucleic acid sequence having at
least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO: 116. In some of any such embodiments, the scFv is or
contains the amino acid sequence encoded by SEQ ID NO: 117. In some
of any such embodiments, the scFv is or contains the amino acid
sequence encoded by SEQ ID NO: 125 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 125. In some of any such embodiments, the
scFv is or contains the amino acid sequence encoded by SEQ ID NO:
126.
[0059] In some of any such embodiments, the anti-ROR1 chimeric
antigen receptor further contains a spacer between the
extracellular antigen-binding domain and the transmembrane domain.
In some of any such embodiments, the spacer contains at least a
portion of an immunoglobulin or a variant thereof. In some of any
such embodiments, the spacer contains at least a portion of a hinge
region of an immunoglobulin or a variant thereof. In some of any
such embodiments, the spacer is less than at or about 15 amino
acids in length. In some of any such embodiments, the spacer is or
contains an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:1, 26, 27, 29, 31, 32, 33 or 135.
[0060] In some of any such embodiments, the at least a portion of a
hinge region contains all or a portion of an IgG4 hinge region. In
some of any such embodiments, the at least a portion of a hinge
region contains all or a portion of a human IgG4 hinge region, or a
variant thereof. In some of any such embodiments, the at least a
portion of a hinge region contains all or a portion of an IgG2
hinge region. In some of any such embodiments, the at least a
portion of a hinge region contains all or a portion of a human IgG2
hinge region, or a variant thereof.
[0061] In some of any such embodiments, the spacer is or contains
the sequence set forth in SEQ ID NO:1. In some of any such
embodiments, the spacer is or contains the amino acid sequence
encoded by SEQ ID NO: 2 or 30 or a nucleic acid sequence having at
least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO: 2 or 30. In some of any such embodiments, the spacer is or
contains the amino acid sequence encoded by SEQ ID NO: 30.
[0062] In some of any such embodiments, the spacer is or contains
the sequence set forth in SEQ ID NO:135. In some of any such
embodiments, the spacer is or contains the amino acid sequence
encoded by SEQ ID NO: 192 or 136 or a nucleic acid sequence having
at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID NO: 192 or 136. In some of any such embodiments, the spacer
is or contains the amino acid sequence encoded by SEQ ID NO:
136.
[0063] In some of any such embodiments, the spacer contains at
least a portion of a C.sub.H3 region of an immunoglobulin or a
variant thereof. In some of any such embodiments, the at least a
portion of a C.sub.H3 region contains all or a portion of an IgG4
C.sub.H3. In some of any such embodiments, the at least a portion
of a C.sub.H3 region contains all or a portion of an IgG2 C.sub.H3.
In some of any such embodiments, the at least a portion of a
C.sub.H3 region contains all or a portion of an IgG4 C.sub.H3
and/or an IgG2 C.sub.H3. In some of any such embodiments, the IgG4
C.sub.H3 is a human IgG4 C.sub.H3 and the IgG2 C.sub.H3 is a human
IgG2 C.sub.H3. In some of any such embodiments, the spacer contains
at least a portion of a hinge region and at least a portion of a
C.sub.H3 region of an immunoglobulin or a variant thereof. In some
of any such embodiments, the spacer is at or about 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 or 125 amino
acids in length, or has a length between any of the foregoing. In
some of any such embodiments, the spacer is at or about 120 amino
acids in length.
[0064] In some of any such embodiments, the spacer is or contains
an amino acid sequence having at least at or about 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 138. In some of any such embodiments, the
spacer is or contains the sequence set forth in SEQ ID NO:138. In
some of any such embodiments, the spacer is or contains the amino
acid sequence encoded by SEQ ID NO: 193 or 139 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 193 or 139. In some of any such
embodiments, the spacer is or contains the amino acid sequence
encoded by SEQ ID NO: 139.
[0065] In some of any such embodiments, the spacer is at or about
120 amino acids in length. In some of any such embodiments, the
spacer is or contains an amino acid sequence having at least at or
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity to SEQ ID NO: 3. In some of any such
embodiments, the spacer is or contains the sequence set forth in
SEQ ID NO:3. In some of any such embodiments, the spacer is or
contains the amino acid sequence encoded by SEQ ID NO: 4 or 137 or
a nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 4 or 137. In some of
any such embodiments, the spacer is or contains the amino acid
sequence encoded by SEQ ID NO: 137.
[0066] In some of any such embodiments, the spacer contains at
least a portion of a C.sub.H2 of an immunoglobulin or a variant
thereof. In some of any such embodiments, the at least a portion of
a C.sub.H2 region contains all or a portion of an IgG4 C.sub.H2. In
some of any such embodiments, the at least a portion of a C.sub.H2
region contains all or a portion of an IgG2 C.sub.H2. In some of
any such embodiments, the at least a portion of a C.sub.H2 region
contains all or a portion of an IgG4 C.sub.H2 and/or an IgG2
C.sub.H2. In some of any such embodiments, the IgG4 C.sub.H2 is a
human IgG4 C.sub.H2 and the IgG2 C.sub.H2 is a human IgG2 C.sub.H2.
In some of any such embodiments, the spacer contains at least a
portion of a hinge region, at least a portion of a C.sub.H2 and at
least a portion of a C.sub.H3 region of an immunoglobulin or a
variant thereof. In some of any such embodiments, the spacer is at
or about 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221,
222, 223, 224, 225, 226, 227, 228, 229 or 230 amino acids in
length, or has a length between any of the foregoing. In some of
any such embodiments, one or more of the hinge region, the C.sub.H2
region and the C.sub.H3 region contains all or a portion of a
C.sub.H2 region and all or a portion of a C.sub.H3 region from
human IgG4. In some of any such embodiments, one or more of the
hinge region, the C.sub.H2 region and the C.sub.H3 region is
chimeric and contains a hinge, a C.sub.H2 region and a C.sub.H3
region from human IgG4 and human IgG2.
[0067] In some of any such embodiments, the spacer contains a
IgG4/2 chimeric hinge region or a modified IgG4 hinge region
containing at least one amino acid replacement compared to a human
IgG4 hinge; an IgG2/4 chimeric C.sub.H2 region; and an IgG4
C.sub.H3 region.
[0068] In some of any such embodiments, the spacer is or contains
an amino acid sequence having at least at or about 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO:194. In some of any such embodiments, the
spacer is or contains the sequence set forth in SEQ ID NO: 194. In
some of any such embodiments, the spacer is or contains the amino
acid sequence encoded by SEQ ID NO: 195 or 196 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 195 or 196. In some of any such
embodiments, the spacer is or contains the amino acid sequence
encoded by SEQ ID NO: 196.
[0069] In some of any such embodiments, the spacer is or contains
an amino acid sequence having at least at or about 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO:37. In some of any such embodiments, the
spacer is or contains the sequence set forth in SEQ ID NO: 37. In
some of any such embodiments, the spacer is or contains the amino
acid sequence encoded by SEQ ID NO: 38 or 140 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 38 or 140. In some of any such
embodiments, the spacer is or contains the amino acid sequence
encoded by SEQ ID NO: 140.
[0070] In some of any such embodiments, the intracellular signaling
region contains an intracellular signaling domain. In some of any
such embodiments, the intracellular signaling domain is capable of
inducing a primary activation signal in a T cell, is a T cell
receptor (TCR) component or contains an immunoreceptor
tyrosine-based activation motif (ITAM). In some of any such
embodiments, the intracellular signaling domain is or contains a
cytoplasmic signaling domain of a CD3-zeta (CD3.zeta.) chain or a
functional variant or signaling portion thereof. In some of any
such embodiments, the intracellular signaling domain is human or is
from a human protein. In some of any such embodiments, the
intracellular signaling domain is or contains the sequence set
forth in SEQ ID NO:13, 14 or 15, or an amino acid sequence having
at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% sequence identity to SEQ ID NO:13, 14 or 15. In some of any
such embodiments, the intracellular signaling domain is or contains
the sequence set forth in SEQ ID NO:13.
[0071] In some of any such embodiments, the intracellular signaling
region further contains a costimulatory signaling region. In some
of any such embodiments, the costimulatory signaling region
contains an intracellular signaling domain of a T cell
costimulatory molecule or a signaling portion thereof. In some of
any such embodiments, the costimulatory signaling region contains
an intracellular signaling domain of CD28, 4-1BB, or ICOS, or a
signaling portion thereof. In some of any such embodiments, the
costimulatory signaling region is human or is from a human protein.
In some of any such embodiments, the costimulatory signaling region
contains an intracellular signaling domain of CD28. In some of any
such embodiments, the costimulatory signaling region is or contains
the sequence set forth in SEQ ID NO:10 or an amino acid sequence
having at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% sequence identity to SEQ ID NO:10. In some of any such
embodiments, the costimulatory signaling region contains an
intracellular signaling domain of 4-1BB. In some of any such
embodiments, the costimulatory signaling region is or contains the
sequence set forth in SEQ ID NO:12 or an amino acid sequence having
at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% sequence identity to SEQ ID NO: 12.
[0072] In some of any such embodiments, the anti-ROR1 chimeric
antigen receptor further contains a transmembrane region. In some
of any such embodiments, the costimulatory signaling region is
between the transmembrane region and the intracellular signaling
domain. In some of any such embodiments, the transmembrane region
is or contains a transmembrane domain from CD4, CD28, or CD8. In
some of any such embodiments, the transmembrane region is or
contains a transmembrane domain from CD28. In some of any such
embodiments, the transmembrane region is human or is from a human
protein.
[0073] In some of any such embodiments, the transmembrane domain is
or contains SEQ ID NO: 8 or an amino acid sequence having at least
at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
sequence identity to SEQ ID NO: 8. In some of any such embodiments,
the transmembrane domain is or contains the sequence set forth in
SEQ ID NO: 8. In some of any such embodiments, the transmembrane
domain is or contains the amino acid sequence encoded by SEQ ID NO:
197 or 198 or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 197 or
198. In some of any such embodiments, the transmembrane domain is
or contains the amino acid sequence encoded by SEQ ID NO: 198.
[0074] In some of any such embodiments, the transmembrane domain is
or contains SEQ ID NO: 149 or an amino acid sequence having at
least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% sequence identity to SEQ ID NO: 149. In some of any such
embodiments, the transmembrane domain is or contains the sequence
set forth in SEQ ID NO: 149. In some of any such embodiments, the
transmembrane domain is or contains the amino acid sequence encoded
by SEQ ID NO: 147 or 148 or a nucleic acid sequence having at least
at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 147 or 148. In some of any such embodiments, the transmembrane
domain is or contains the amino acid sequence encoded by SEQ ID NO:
148.
[0075] In some of any such embodiments, the encoded chimeric
antigen receptor contains from its N to C terminus in order: the
extracellular antigen-binding domain, the spacer, the transmembrane
region and the intracellular signaling region. In some of any such
embodiments, the encoded chimeric antigen receptor contains, from
its N to C terminus in order: an extracellular antigen-binding
domain containing an scFv, a spacer containing a modified IgG4
hinge; a transmembrane domain; and an intracellular signaling
region containing a cytoplasmic signaling domain of a CD3-zeta
(CD3.zeta.) chain and an intracellular signaling domain of a
costimulatory signaling region. In some of any such embodiments,
the encoded chimeric antigen receptor contains, from its N to C
terminus in order: an extracellular antigen-binding domain
containing an scFv, a spacer containing a modified IgG4 hinge
containing the sequence set forth in SEQ ID NO:135; a transmembrane
domain from a human CD28; and an intracellular signaling region
containing a cytoplasmic signaling domain of a CD3-zeta (CD3.zeta.)
chain and an intracellular signaling domain of a costimulatory
signaling region containing an intracellular signaling domain of
4-1BB. In some of any such embodiments, the encoded chimeric
antigen receptor contains, from its N to C terminus in order: an
extracellular antigen-binding domain containing an scFv, a spacer
containing a modified IgG4 hinge-C.sub.H3; a transmembrane domain;
and an intracellular signaling region containing a cytoplasmic
signaling domain of a CD3-zeta (CD3.zeta.) chain and an
intracellular signaling domain of a costimulatory signaling region.
In some of any such embodiments, the encoded chimeric antigen
receptor contains, from its N to C terminus in order: an
extracellular antigen-binding domain containing an scFv, a spacer
containing a modified IgG4 hinge-C.sub.H3 containing the sequence
set forth in SEQ ID NO: 138; a transmembrane domain from a human
CD28; and an intracellular signaling region containing a
cytoplasmic signaling domain of a CD3-zeta (CD3.zeta.) chain and an
intracellular signaling domain of a costimulatory signaling region
containing an intracellular signaling domain of 4-1BB. In some of
any such embodiments, the extracellular antigen-binding domain is
an scFv.
[0076] In some of any such embodiments, the anti-ROR1 chimeric
antigen receptor is or contains the sequence set forth in SEQ ID
NO: 184, 185, 186, 187, 188 or 189 or a sequence that exhibits at
least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% sequence identity to the sequence set forth in SEQ
ID NO: 184, 185, 186, 187, 188 or 189.
[0077] In some of any such embodiments, the anti-ROR1 chimeric
antigen receptor is or contains the sequence set forth in SEQ ID
NO: 184 or a sequence that exhibits at least 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the sequence set forth in SEQ ID NO: 184. In some of
any such embodiments, the anti-ROR1 chimeric antigen receptor is or
contains the sequence set forth in SEQ ID NO: 184. In some of any
such embodiments, the anti-ROR1 chimeric antigen receptor is or
contains the sequence set forth in SEQ ID NO: 185 or a sequence
that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence
set forth in SEQ ID NO:185. In some of any such embodiments, the
anti-ROR1 chimeric antigen receptor is or contains the sequence set
forth in SEQ ID NO: 185. In some of any embodiments, the anti-ROR1
chimeric antigen receptor is or comprises the sequence set forth in
SEQ ID NO: 186 or a sequence that exhibits at least 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the sequence set forth in SEQ ID NO: 186. In
some of any embodiments, the anti-ROR1 chimeric antigen receptor is
or comprises the sequence set forth in SEQ ID NO: 186. In some of
any embodiments, the anti-ROR1 chimeric antigen receptor is or
comprises the sequence set forth in SEQ ID NO: 187 or a sequence
that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence
set forth in SEQ ID NO:187. In some of any embodiments, the
anti-ROR1 chimeric antigen receptor is or comprises the sequence
set forth in SEQ ID NO: 187. In some of any embodiments, the
anti-ROR1 chimeric antigen receptor is or comprises the sequence
set forth in SEQ ID NO: 188 or a sequence that exhibits at least
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the sequence set forth in SEQ ID
NO: 188. In some of any embodiments, the anti-ROR1 chimeric antigen
receptor is or comprises the sequence set forth in SEQ ID NO: 188.
In some of any embodiments, the anti-ROR1 chimeric antigen receptor
is or comprises the sequence set forth in SEQ ID NO: 189 or a
sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
sequence set forth in SEQ ID NO:189. In some of any embodiments,
the anti-ROR1 chimeric antigen receptor is or comprises the
sequence set forth in SEQ ID NO: 189.
[0078] In some of any such embodiments, the anti-ROR1 chimeric
antigen receptor is encoded by the sequence set forth in SEQ ID NO:
156, 157, 158, 159, 160 or 161 or a sequence that exhibits at least
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
sequence set forth in SEQ ID NO: 156, 157, 158, 159, 160 or 161. In
some of any such embodiments, the anti-ROR1 chimeric antigen
receptor is encoded by the sequence set forth in SEQ ID NO: 156 or
a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% sequence identity to the sequence set forth in SEQ ID NO: 156.
In some of any such embodiments, the anti-ROR1 chimeric antigen
receptor is encoded by the sequence set forth in SEQ ID NO: 156. In
some of any such embodiments, the anti-ROR1 chimeric antigen
receptor is encoded by the sequence set forth in SEQ ID NO: 157 or
a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% sequence identity to the sequence set forth in SEQ ID NO: 157.
In some of any such embodiments, the anti-ROR1 chimeric antigen
receptor is encoded by the sequence set forth in SEQ ID NO:
157.
[0079] In some of any such embodiments, said anti-ROR1 chimeric
antigen receptor specifically binds to a receptor tyrosine
kinase-like orphan receptor 1 (ROR1) protein. In some of any such
embodiments, said anti-ROR1 chimeric antigen receptor specifically
binds to a human ROR1 protein. In some of any such embodiments, the
human ROR1 protein contains an amino acid sequence set forth in SEQ
ID NO: 144, 145 or 146. In some of any such embodiments, said
anti-ROR1 chimeric antigen receptor does not bind to, is not
cross-reactive to, or binds at a lower level or degree or affinity
to a receptor tyrosine kinase-like orphan receptor 2 (ROR2)
protein. In some of any such embodiments, said anti-ROR1 chimeric
antigen receptor does not bind to, is not cross-reactive to, or
binds at a lower level or degree or affinity to a human ROR2.
[0080] In some of any such embodiments, the extent, level or degree
or affinity of binding of said anti-ROR1 chimeric antigen receptor
to a human ROR2 is at least at or about 75%, 80%, 90%, 95% or 99%
less than the extent, level or degree or affinity of binding to a
human ROR1. In some of any such embodiments, binding is compared
under the same or substantially the same conditions or assay. In
some of any such embodiments, said anti-ROR1 chimeric antigen
receptor exhibits the same, substantially the same or lower level
or degree of signaling or activity in the presence of a ROR2
protein, compared to the level or degree of signaling or activity
in the presence of a ROR1 protein. In some of any such embodiments,
said anti-ROR1 chimeric antigen receptor exhibits the same,
substantially the same or lower level or degree of signaling or
activity in the presence of a human ROR2 protein, compared to the
level or degree of signaling or activity in the presence of a human
ROR1 protein. In some of any such embodiments, activity is compared
under the same or substantially the same conditions or assay. In
some of any such embodiments, said anti-ROR1 chimeric antigen
receptor exhibits a level or degree of signaling or activity in the
presence of a human ROR2 that is at least at or about 75%, 80%,
90%, 95% or 99% less than the level or degree of signaling or
activity in the presence of a human ROR1. In some of any such
embodiments, activity is compared under the same or substantially
the same conditions or assay.
[0081] In some of any such embodiments, said anti-ROR1 chimeric
antigen receptor exhibits the same, substantially the same or lower
level or degree of signaling or activity in the presence of a ROR2
protein compared to a reference ROR1-specific chimeric antigen
receptor. In some of any such embodiments, said anti-ROR1 chimeric
antigen receptor exhibits the same, substantially the same or lower
level or degree of signaling or activity in the presence of a human
ROR2, compared to a reference ROR1-specific chimeric antigen
receptor. In some of any embodiments, activity is compared under
the same or substantially the same conditions or assay.
[0082] In some of any such embodiments, said anti-ROR1 chimeric
antigen receptor exhibits the same, substantially the same or lower
level or degree of signaling or activity in the presence of a ROR2
protein, compared to a reference ROR1-specific chimeric antigen
receptor. In some of any such embodiments, said anti-ROR1 chimeric
antigen receptor exhibits the same, substantially the same or lower
level or degree of signaling or activity in the presence of a human
ROR2 protein, compared to a reference ROR1-specific chimeric
antigen receptor. In some of any such embodiments, activity is
compared under the same or substantially the same conditions or
assay. In some of any such embodiments, said anti-ROR1 chimeric
antigen receptor exhibits the same, substantially the same or
higher antigen-specific signaling or antigen dependent activity or
signaling compared to a reference ROR1-specific chimeric antigen
receptor. In some of any such embodiments, activity is compared
under the same or substantially the same conditions or assay. In
some of any such embodiments, said anti-ROR1 chimeric antigen
receptor exhibits the same, substantially the same or lower tonic
signaling or antigen independent activity or signaling compared to
a reference ROR1-specific chimeric antigen receptor. In some of any
such embodiments, activity is compared under the same or
substantially the same conditions or assay. In some of any such
embodiments, said anti-ROR1 chimeric antigen receptor exhibits a
level or degree of tonic signaling or antigen independent activity
or signaling that is at least at or about 75%, 80%, 90%, 95% or 99%
less than the level or degree of tonic signaling or antigen
independent activity of a reference ROR1-specific chimeric antigen
receptor. In some of any such embodiments, activity is compared
under the same or substantially the same conditions or assay.
[0083] In some of any such embodiments, the reference ROR1-specific
chimeric antigen receptor contains the anti-ROR1 antibody R12 or
the anti-ROR1 antibody 2A2 or an antigen-binding fragment thereof.
In some of any such embodiments, the reference ROR1-specific
chimeric antigen receptor contains an scFv from R12 or 2A2. In some
of any such embodiments, the reference ROR1-specific chimeric
antigen receptor contains the anti-ROR1 antibody R12 or an scFv
from R12 or 2A2.
[0084] Also provided herein is a polynucleotide containing a
nucleic acid encoding all or a portion of the anti-ROR1 antibody or
antigen-binding domain thereof of any of the embodiments provided
herein, the single chain cell surface protein of any of the
embodiments provided herein, the conjugate of any of the
embodiments provided herein, or the anti-ROR1 chimeric antigen
receptor of any of the embodiments provided herein.
[0085] In some of any such embodiments, said polynucleotide
contains a nucleic acid encoding the V.sub.H containing the
sequence set forth in SEQ ID NO: 110, 119, 101 or 128 or a nucleic
acid sequence having at least at or about 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 110, 119, 101 or 128, and a
nucleic acid encoding the V.sub.L containing the sequence set forth
in SEQ ID NO: 113, 122 or 104, or a nucleic acid sequence having at
least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO: 113, 122 or 104. In some of any such embodiments, said
polynucleotide contains a nucleic acid encoding the V.sub.H
containing the sequence set forth in SEQ ID NO: 110 or a nucleic
acid sequence having at least at or about 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 110, and a nucleic acid encoding
the V.sub.L containing the sequence set forth in SEQ ID NO: 113, or
a nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 113. In some of any
such embodiments, said polynucleotide contains a nucleic acid
encoding the V.sub.H containing the sequence set forth in SEQ ID
NO: 119 or a nucleic acid sequence having at least at or about 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 119, and a
nucleic acid encoding the V.sub.L containing the sequence set forth
in SEQ ID NO: 122, or a nucleic acid sequence having at least at or
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:
122.
[0086] In some of any such embodiments, said polynucleotide
contains a nucleic acid encoding the scFv containing the sequence
set forth in SEQ ID NO: 116, 125, 107 or 132 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 116, 125, 107 or 132. In some of any
such embodiments, said polynucleotide contains a nucleic acid
encoding the scFv containing the sequence set forth in SEQ ID NO:
116 or a nucleic acid sequence having at least at or about 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 116. In some
of any such embodiments, said polynucleotide contains a nucleic
acid encoding the scFv containing the sequence set forth in SEQ ID
NO: 125 or a nucleic acid sequence having at least at or about 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 125.
[0087] In some of any such embodiments, said polynucleotide
contains a nucleic acid encoding the spacer containing the sequence
set forth in SEQ ID NO: 192 or a nucleic acid sequence having at
least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO: 192. In some of any such embodiments, said polynucleotide
contains a nucleic acid encoding the spacer containing the sequence
set forth in SEQ ID NO: 193 or a nucleic acid sequence having at
least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO: 193. In some of any such embodiments, said polynucleotide
contains a nucleic acid encoding the spacer containing the sequence
set forth in SEQ ID NO: 195 or a nucleic acid sequence having at
least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO: 195.
[0088] In some of any such embodiments, the nucleic acid encoding
the anti-ROR1 antibody or antigen-binding domain thereof, the
single chain cell surface protein, the conjugate or the anti-ROR1
chimeric antigen receptor contains at least one modified splice
donor or splice acceptor site or both, said modified splice donor
and/or acceptor site containing one or more nucleotide
modifications corresponding to a reference splice donor site and/or
reference splice acceptor site. In some of any such embodiments,
the one or more nucleotide modifications contain a nucleic acid
substitution. In some of any such embodiments, the reference splice
donor and/or reference splice acceptor sites are canonical,
non-canonical, or cryptic splice sites. In some of any embodiments,
the polynucleotide is optimized by splice site elimination.
[0089] In some of any such embodiments: the reference splice donor
or reference splice acceptor site(s) or both has a splice site
prediction score of at least at or about 0.4, 0.5, 0.6, 0.70, 0.75,
0.80, 0.85, 0.90, 0.95, 0.99 or 1.0; or the reference splice donor
or reference splice acceptor site(s) or both is/are predicted to be
involved in a splice event with a probability of at least at or
about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
[0090] In some of any such embodiments: the reference splice donor
or reference splice acceptor site(s) or both has a splice site
prediction score of at least at or about 0.70, 0.75, 0.80, 0.85,
0.90, 0.95, 0.99 or 1.0; and/or the reference splice donor or
reference splice acceptor site(s) or both is/are predicted to be
involved in a splice event with a probability of at least at or
about 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
[0091] In some of any such embodiments, at least one of the one or
more nucleotide modifications are within 1, 2, 3, 4, 5, 6, 7, 8, 9
or 10 residues of the splice site junction of the reference splice
acceptor and/or reference splice donor site. In some of any such
embodiments, the one or more nucleotide modifications is silent or
results in a degenerate codon or does not change the amino acid
sequence of the encoded protein, or one or more or all of the
foregoing.
[0092] In some of any such embodiments, upon expression of the
polynucleotide in a cell, the transcribed RNA from the
polynucleotide, exhibits at least at or about 70%, at or about 75%,
at or about 80%, at or about 85%, at or about 90%, or at or about
95% RNA homogeneity. In some of any such embodiments, upon
expression in a cell, the transcribed RNA from the polynucleotide
exhibits reduced heterogeneity compared to the heterogeneity of the
mRNA transcribed from a reference polynucleotide, said reference
polynucleotide encoding the same amino acid sequence as the
polynucleotide, wherein the reference polynucleotide differs by the
presence of one or more splice donor site or one or more splice
acceptor site or both, in the nucleic acid encoding the spacer or
contains one or more nucleotide modifications compared to the
polynucleotide. In some of any such embodiments, the RNA
heterogeneity is reduced by greater than at or about 10%, 15%, 20%,
25%, 30%, 40% or 50% or more. In some of any such embodiments, the
transcribed RNA from the reference polynucleotide exhibits greater
than at or about 10%, 15%, 20%, 25%, 30%, 40% or 50% or more RNA
heterogeneity. In some of any such embodiments, the transcribed RNA
messenger RNA (mRNA). In some of any such embodiments, the RNA
homogeneity or heterogeneity or both is determined by agarose gel
electrophoresis, chip-based capillary electrophoresis, analytical
ultracentrifugation, field flow fractionation, or liquid
chromatography.
[0093] In some of any such embodiments, the polynucleotide is
codon-optimized for expression in a human cell.
[0094] In some of any such embodiments, said polynucleotide
contains a nucleic acid encoding the V.sub.H containing the
sequence set forth in SEQ ID NO: 111, 120, 102 or 129, and a
nucleic acid encoding the V.sub.L containing the sequence set forth
in SEQ ID NO: 114, 123, 105 or 131. In some of any such
embodiments, said polynucleotide contains a nucleic acid encoding
the V.sub.H containing the sequence set forth in SEQ ID NO: 111,
and a nucleic acid encoding the V.sub.L containing the sequence set
forth in SEQ ID NO: 114. In some of any such embodiments, said
polynucleotide contains a nucleic acid encoding the V.sub.H
containing the sequence set forth in SEQ ID NO: 120, and a nucleic
acid encoding the V.sub.L containing the sequence set forth in SEQ
ID NO: 123.
[0095] In some of any such embodiments, said polynucleotide
contains a nucleic acid encoding the scFv containing the sequence
set forth in SEQ ID NO: 117, 126, 108 or 133. In some of any such
embodiments, said polynucleotide contains a nucleic acid encoding
the scFv containing the sequence set forth in SEQ ID NO: 117. In
some of any such embodiments, said polynucleotide contains a
nucleic acid encoding the scFv containing the sequence set forth in
SEQ ID NO: 126.
[0096] In some of any such embodiments, said polynucleotide
contains a nucleic acid encoding the spacer containing the sequence
set forth in SEQ ID NO:136. In some of any such embodiments, said
polynucleotide contains a nucleic acid encoding the spacer
containing the sequence set forth in SEQ ID NO:139. In some of any
such embodiments, said polynucleotide contains a nucleic acid
encoding the spacer containing the sequence set forth in SEQ ID
NO:196.
[0097] In some of any such embodiments, said polynucleotide
contains the sequence set forth in SEQ ID NO: 156, 157, 158, 159,
160 or 161 or a sequence that exhibits at least 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% sequence identity to the sequence set forth in SEQ
ID NO: 156, 157, 158, 159, 160 or 161. In some of any such
embodiments, said polynucleotide contains the sequence set forth in
SEQ ID NO: 156 or a sequence that exhibits at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% sequence identity to the sequence set forth
in SEQ ID NO: 156. In some of any such embodiments, said
polynucleotide contains the sequence set forth in SEQ ID NO: 156.
In some of any such embodiments, said polynucleotide contains the
sequence set forth in SEQ ID NO: 157 or a sequence that exhibits at
least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
sequence set forth in SEQ ID NO: 157. In some of any such
embodiments, said polynucleotide contains the sequence set forth in
SEQ ID NO: 157.
[0098] In some of any such embodiments, the polynucleotide further
contains a CD33 signal sequence, a GM-CSF signal sequence, a CD8
signal sequence or an Ig kappa signal sequence. In some of any such
embodiments, the polynucleotide further contains a CD33 signal
sequence. In some of any such embodiments, the CD33 signal sequence
is set forth in SEQ ID NO:190 or a nucleic acid sequence having at
least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO:190.
[0099] Also provided herein is a vector, containing the
polynucleotide of any of the embodiments provided herein. In some
embodiments, the vector is a viral vector. In some embodiments, the
viral vector is a retroviral vector or a lentiviral vector.
[0100] Also provided herein is a cell containing the anti-ROR1
chimeric antigen receptor of any of the embodiments provided
herein. Also provided herein is a cell containing the
polynucleotide of any of the embodiments provided herein, or the
vector of any of the embodiments provided herein. Also provided
herein is a cell containing the anti-ROR1 antibody or
antigen-binding fragment thereof of the embodiments provided
herein, the conjugate of any of the embodiments provided herein,
the single chain cell surface protein of any of the embodiments
provided herein, the anti-ROR1 chimeric antigen receptor of any of
the embodiments provided herein, the polynucleotide of any of the
embodiments provided herein, or the vector of any of the
embodiments provided herein.
[0101] In some embodiments, the cell is a lymphocyte. In some
embodiments, the cell is an NK cell or a T cell. In some of any
such embodiments, the cell is a T cell and the T cell is a CD4+ or
a CD8+ T cell. In some of any such embodiments, the cell is a
primary cell obtained from a subject.
[0102] In some of any such embodiments, among a plurality of the
cells, less than at or about 10%, at or about 9%, at or about 8%,
at or about 7%, at or about 5%, at or about 4%, at or about 3%, at
or about 2% or at or about 1% of the cells in the plurality contain
an anti-ROR1 chimeric antigen receptor that exhibits tonic
signaling or antigen independent activity or signaling.
[0103] Also provided herein is a composition containing the
anti-ROR1 antibody or antigen-binding fragment thereof of any of
the embodiments provided herein, the conjugate of any of the
embodiments provided herein, the single chain cell surface protein
of any of the embodiments provided herein or the anti-ROR1 chimeric
antigen receptor of any of the embodiments provided herein.
[0104] Also provided herein is a composition containing the cell of
any of the embodiments provided herein. In some of any such
embodiments, the composition further contains a pharmaceutically
acceptable excipient. In some of any such embodiments, the
composition contains CD4+ and CD8+ T cells and the ratio of CD4+ to
CD8+ T cells is from at or about 1:3 to 3:1. In some of any such
embodiments, the composition contains CD4+ and CD8+ T cells and the
ratio of CD4+ to CD8+ T cells is at or about 1:2 to 2:1. In some of
any such embodiments, the composition contains CD4+ and CD8+ T
cells and the ratio of CD4+ to CD8+ T cells is at or about 1:1.
[0105] In some of any such embodiments, among a plurality of the
cells in the composition, less than at or about 10%, at or about
9%, at or about 8%, at or about 7%, at or about 5%, at or about 4%,
at or about 3%, at or about 2% or at or about 1% of the cells in
the plurality contain an anti-ROR1 chimeric antigen receptor that
exhibits tonic signaling or antigen independent activity or
signaling.
[0106] Also provided herein is a method of treatment, containing
administering the composition of any of the embodiments provided
herein to a subject having a disease or disorder associated with
ROR1.
[0107] Also provided herein is a method of treatment, containing
administering the anti-ROR1 antibody or antigen-binding fragment
thereof of any of the embodiments provided herein, the conjugate of
any of the embodiments provided herein, the single chain cell
surface protein of any of the embodiments provided herein, the
anti-ROR1 chimeric antigen receptor of any of the embodiments
provided herein, the polynucleotide of any of the embodiments
provided herein, the vector of any of the embodiments provided
herein, or the cell of any of the embodiments provided herein to a
subject having a disease or disorder associated with ROR1. Also
provided herein is a composition of any of the embodiments provided
herein for use in treating a disease or disorder associated with
ROR1. Also provided herein is a use of a composition of any of the
embodiments provided herein for the manufacture of a medicament for
treating a disease or disorder associated with ROR1. Also provided
herein is a use of a composition of any of the embodiments provided
herein for the treatment of a disease or disorder associated with
ROR1. Also provided herein is a method of treatment, containing
administering the cells of any of the embodiments provided herein
to a subject having a disease or disorder associated with ROR1.
[0108] Also provided herein is a method of treatment, containing
administering the anti-ROR1 antibody or antigen-binding fragment
thereof of any of the embodiments provided herein, the conjugate of
any of the embodiments provided herein, the single chain cell
surface protein of any of the embodiments provided herein, the
anti-ROR1 chimeric antigen receptor of any of the embodiments
provided herein, the polynucleotide of any of the embodiments
provided herein, the vector of any of the embodiments provided
herein, or the cell of any of the embodiments provided herein to a
subject having a disease or disorder associated with ROR1. Also
provided herein is a cell of any of the embodiments provided herein
for use in treating a disease or disorder associated with ROR1.
Also provided herein is a use of a cell of any of the embodiments
provided herein for the manufacture of a medicament for treating a
disease or disorder associated with ROR1. Also provided herein is a
use of a cell of any of the embodiments provided herein for the
treatment of a disease or disorder associated with ROR1.
[0109] In some of any of the provided embodiments, the disease or
disorder associated with ROR1 is a cancer. In some of any of the
provided embodiments, the cancer is a ROR1-expressing cancer. In
some of any of the provided embodiments, the cancer is associated
with a ROR1-expressing solid tumor or a ROR1-expressing hematologic
malignancy.
[0110] In some of any of the provided embodiments, the cancer is
associated with a ROR1-expressing solid tumor. In some of any of
the provided embodiments, the cancer associated with a solid tumor
is selected from the group consisting of neuroblastoma, renal cell
carcinoma, colon cancer, colorectal cancer, breast cancer,
epithelial squamous cell cancer, melanoma, myeloma, stomach cancer,
brain cancer, lung cancer, pancreatic cancer, cervical cancer,
ovarian cancer, liver cancer, bladder cancer, prostate cancer,
testicular cancer, thyroid cancer, uterine cancer, adrenal cancer
and head and neck cancer. In some of any of the provided
embodiments, the lung cancer is a non-small cell lung cancer
(NSCLC), lung adenocarcinoma, adenocarcinoma, squamous cell
carcinoma, small cell carcinoma, and atypical carcinoid. In some of
any of the provided embodiments, the lung cancer is a non-small
cell lung cancer (NSCLC). In some of any of the provided
embodiments, the breast cancer is a triple negative breast cancer
(TNBC).
[0111] In some of any of the provided embodiments, the cancer is
associated with a ROR1-expressing hematologic malignancy. In some
of any of the provided embodiments, the hematologic malignancy is
selected from the group consisting of B cell leukemia, lymphoma, B
cell chronic lymphocytic leukemia (CLL), acute myeloid leukemia
(AML), acute lymphocytic leukemia (ALL), Burkitt's Lymphoma or
mantle cell lymphoma (MCL).
[0112] In some embodiments of the methods provided herein, the
disease or disorder associated with ROR1 is a cancer. In some
embodiments of the methods provided herein, the cancer is a
ROR1-expressing cancer. In some embodiments of the methods provided
herein, the cancer is associated with a ROR1-expressing solid tumor
or a ROR1-expressing hematologic malignancy.
[0113] In some embodiments of the methods provided herein, the
cancer is associated with a ROR1-expressing solid tumor. In some
embodiments of the methods provided herein, the cancer associated
with a solid tumor is selected from the group consisting of
neuroblastoma, renal cell carcinoma, colon cancer, colorectal
cancer, breast cancer, epithelial squamous cell cancer, melanoma,
myeloma, stomach cancer, brain cancer, lung cancer, pancreatic
cancer, cervical cancer, ovarian cancer, liver cancer, bladder
cancer, prostate cancer, testicular cancer, thyroid cancer, uterine
cancer, adrenal cancer and head and neck cancer. In some
embodiments of the methods provided herein, the lung cancer is a
non-small cell lung cancer (NSCLC), lung adenocarcinoma,
adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and
atypical carcinoid. In some embodiments of the methods provided
herein, the lung cancer is a non-small cell lung cancer (NSCLC). In
some embodiments of the methods provided herein, the breast cancer
is a triple negative breast cancer (TNBC).
[0114] In some embodiments of the methods provided herein, the
cancer is associated with a ROR1-expressing hematologic malignancy.
In some embodiments of the methods provided herein, the hematologic
malignancy is selected from the group consisting of B cell
leukemia, lymphoma, B cell chronic lymphocytic leukemia (CLL),
acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL),
Burkitt's Lymphoma or mantle cell lymphoma (MCL).
[0115] In some embodiments of the cells or compositions for use
provided herein, the disease or disorder associated with ROR1 is a
cancer. In some embodiments of the cells or compositions for use
provided herein, the cancer is a ROR1-expressing cancer. In some
embodiments of the cells or compositions for use provided herein,
the cancer is associated with a ROR1-expressing solid tumor or a
ROR1-expressing hematologic malignancy.
[0116] In some embodiments of the cells or compositions for use
provided herein, the cancer is associated with a ROR1-expressing
solid tumor. In some embodiments of the cells or compositions for
use provided herein, the cancer associated with a solid tumor is
selected from the group consisting of neuroblastoma, renal cell
carcinoma, colon cancer, colorectal cancer, breast cancer,
epithelial squamous cell cancer, melanoma, myeloma, stomach cancer,
brain cancer, lung cancer, pancreatic cancer, cervical cancer,
ovarian cancer, liver cancer, bladder cancer, prostate cancer,
testicular cancer, thyroid cancer, uterine cancer, adrenal cancer
and head and neck cancer. In some embodiments of the cells or
compositions for use provided herein, the lung cancer is a
non-small cell lung cancer (NSCLC), lung adenocarcinoma,
adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and
atypical carcinoid. In some embodiments of the cells or
compositions for use provided herein, the lung cancer is a
non-small cell lung cancer (NSCLC). In some embodiments of the
cells or compositions for use provided herein, the breast cancer is
a triple negative breast cancer (TNBC).
[0117] In some embodiments of the cells or compositions for use
provided herein, the cancer is associated with a ROR1-expressing
hematologic malignancy. In some embodiments of the cells or
compositions for use provided herein, the hematologic malignancy is
selected from the group consisting of B cell leukemia, lymphoma, B
cell chronic lymphocytic leukemia (CLL), acute myeloid leukemia
(AML), acute lymphocytic leukemia (ALL), Burkitt's Lymphoma or
mantle cell lymphoma (MCL).
[0118] In some embodiments of the uses provided herein, the disease
or disorder associated with ROR1 is a cancer. In some embodiments
of the uses provided herein, the cancer is a ROR1-expressing
cancer. In some embodiments of the uses provided herein, the cancer
is associated with a ROR1-expressing solid tumor or a
ROR1-expressing hematologic malignancy.
[0119] In some embodiments of the uses provided herein, the cancer
is associated with a ROR1-expressing solid tumor. In some
embodiments of the uses provided herein, the cancer associated with
a solid tumor is selected from the group consisting of
neuroblastoma, renal cell carcinoma, colon cancer, colorectal
cancer, breast cancer, epithelial squamous cell cancer, melanoma,
myeloma, stomach cancer, brain cancer, lung cancer, pancreatic
cancer, cervical cancer, ovarian cancer, liver cancer, bladder
cancer, prostate cancer, testicular cancer, thyroid cancer, uterine
cancer, adrenal cancer and head and neck cancer. In some
embodiments of the uses provided herein, the lung cancer is a
non-small cell lung cancer (NSCLC). In some embodiments of the uses
provided herein, the breast cancer is a triple negative breast
cancer (TNBC).
[0120] In some embodiments of the uses provided herein, the cancer
is associated with a ROR1-expressing hematologic malignancy. In
some embodiments of the uses provided herein, the hematologic
malignancy is selected from the group consisting of B cell
leukemia, lymphoma, B cell chronic lymphocytic leukemia (CLL),
acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL),
Burkitt's Lymphoma or mantle cell lymphoma (MCL).
[0121] In some of any such embodiments, the disease or disorder
associated with ROR1 is associated with ROR1 expression. In some of
any such embodiments, the disease or disorder associated with ROR1
is a B cell-related disorder. In some of any such embodiments, the
disease or disorder associated with ROR1 is a cancer. In some of
any such embodiments, the cancer is a ROR1-expressing cancer. In
some of any such embodiments, the ROR1-expressing cancer is
selected from the group consisting of B cell leukemia, lymphoma, B
cell chronic lymphocytic leukemia (CLL), acute myeloid leukemia
(AML), acute lymphocytic leukemia (ALL), Burkitt's Lymphoma, mantle
cell lymphoma (MCL), non-small cell lung cancer (NSCLC),
neuroblastoma, renal cell carcinoma, colon cancer, colorectal
cancer, breast cancer, epithelial squamous cell cancer, melanoma,
myeloma, stomach cancer, brain cancer, lung cancer, pancreatic
cancer, cervical cancer, ovarian cancer, liver cancer, bladder
cancer, prostate cancer, testicular cancer, thyroid cancer, uterine
cancer, adrenal cancer and head and neck cancer.
[0122] Also provided herein is a kit containing the anti-ROR1
antibody or antigen-binding fragment thereof of any of the
embodiments provided herein, the single chain cell surface protein
of any of the embodiments provided herein, the conjugate of any of
the embodiments provided herein, the anti-ROR1 chimeric antigen
receptor of any of the embodiments provided herein, the cell of any
of the embodiments provided herein, or the composition of any of
the embodiments provided herein, and instructions for use. In some
of any such embodiments, the instructions are for administering the
anti-ROR1 antibody or antigen-binding fragment thereof, the single
chain cell surface protein, the conjugate, the anti-ROR1 chimeric
antigen receptor, the cell or the composition. In some of any such
embodiments, the instructions are in accord with the method, the
composition for use or the use of any of the embodiments provided
herein.
[0123] Also provided herein is an article of manufacture containing
the anti-ROR1 antibody or antigen-binding fragment thereof of any
of the embodiments provided herein, the single chain cell surface
protein of any of the embodiments provided herein, the conjugate of
any of the embodiments provided herein, the anti-ROR1 chimeric
antigen receptor of any of the embodiments provided herein, the
cell of any of the embodiments provided herein, the composition of
any of the embodiments provided herein, or the kit of any of the
embodiments provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0124] FIG. 1 depicts the relative intracellular TFN-.gamma.,
TNF-.alpha. or IL-2 expression levels by ICS in GFP+ CD4+ T cells
compared to the levels of each cytokine in cells expressing the
reference anti-ROR1 (R12) CAR, after 24 hours stimulation with
plate-bound recombinant ROR1-Fc, in primary T cells expressing
select candidate anti-ROR1 CAR-expressing T cells from a screen as
described in Example 1.
[0125] FIG. 2 shows the relative intracellular TFN-.gamma.,
TNF-.alpha. or IL-2 expression levels by ICS in primary T cells
expressing select candidate anti-ROR1 CAR-expressing T cells from a
screen as described in Example 1, compared to the levels of each
cytokine in cells expressing the reference anti-ROR1 (R12) CAR,
after 24-70 hour co-culture with MDA-MB-231 target cells expressing
ROR1.
[0126] FIGS. 3A-3B depict the results from an in vitro cytotoxicity
assay, after co-culture of NucLight Red (NLR)-labeled H1975 target
cells and primary T cells expressing one of 6 selected candidate
anti-ROR1 CARs, as assessed by measuring the loss of red
fluorescent signal over a period of between 0 and 70 hours. As
controls, cultures of target cells only and co-cultures of mock
cells (not expressing a CAR) with the target cells were
assessed.
[0127] FIGS. 4A-4C show the production of IFN-.gamma., TNF-.alpha.
or IL-2 after 70 hours of co-culture of primary T cells expressing
one of 6 selected candidate anti-ROR1 CARs from two separate
donors, with H1975 (FIGS. 4A and 4B), or the production of IL-2
after co-culture with MDA-MB-231, A549 or BT-549 (FIG. 4C) target
cells. As controls, cultures of target cells only and co-cultures
of mock cells (not expressing a CAR) with the target cells were
assessed.
[0128] FIGS. 5A-5D show the anti-tumor activity, as assessed by the
changes in mean or individual tumor volume after administration of
cells expressing one of 6 selected candidate anti-ROR1 CARs or the
reference anti-ROR1 (R12) CAR, in a H1975 non-small cell lung
cancer (NSCLC) mouse model. FIG. 5A (low dose) and FIG. 5B (high
dose) depict mean tumor volume of all treated mice; in this
depiction, tumor curves were terminated after the first mouse of a
group succumbed to disease. Results from all individual mice are
shown in FIG. 5C (low dose) or FIG. 5D (high dose). As a control,
mice were administered cells not expressing a CAR (mock) or were
untreated.
[0129] FIGS. 6A-6B depict the Kaplan-Meier survival curves after
administration of a low dose (FIG. 6A) or a high dose (FIG. 6B) of
cells expressing one of 6 selected candidate anti-ROR1 CARs or the
reference anti-ROR1 (R12) CAR, in a H1975 non-small cell lung
cancer (NSCLC) mouse model.
[0130] FIGS. 7A-7D show the average number of CD4+ and CD8+
CAR-expressing cells in the blood of each mouse determined at day
10 and day 24 after administration of cells expressing one of 6
selected candidate anti-ROR1 CARs or the reference anti-ROR1 (R12)
CAR, at the low dose (FIGS. 7A and 7B) or high dose (FIGS. 7C and
7D). FIGS. 7E-7F show the average number of CD3+ CAR-expressing
cells in the blood of each mouse determined at day 10 and day 24
after administration of cells expressing one of 6 selected
candidate anti-ROR1 CARs or the reference anti-ROR1 (R12) CAR, at
the low dose (FIG. 7E) or high dose (FIG. 7F). Data are shown as
individual values along with group means.+-.standard deviation.
[0131] FIGS. 8A-8C depict the changes in the mean and individual
tumor volume in H1975 non-small cell lung cancer (NSCLC) mice
administered cells expressing anti-ROR1 CAR-F, CAR-A or the R12
reference CAR, at the low dose (mean: FIG. 8A; individual: FIG. 8C)
or the high dose (mean: FIG. 8B; individual: FIG. 8C). As a
control, mice were administered cells not expressing a CAR (mock)
or were untreated.
[0132] FIGS. 9A-9B depict the Kaplan-Meier survival curves after
administration of a low dose (FIG. 9A) or a high dose (FIG. 9B) of
mice administered cells expressing anti-ROR1 CAR-F, CAR-A or the
R12 reference CAR. As a control, mice were administered cells not
expressing a CAR (mock) or were untreated.
[0133] FIGS. 10A-10D depict the average CD4+ (FIGS. 10A and 10C)
and CD8+ (FIGS. 10B and 10D) CAR+ T cell count per microliter of
blood at days 7, 14 and 21 after administration of a low dose
(FIGS. 10A and 10B) or a high dose (FIGS. 10C and 10D) cells
expressing anti-ROR1 CAR-F, CAR-A or the R12 reference CAR.
[0134] FIGS. 11A-11B depict the number of CD4+ (FIG. 11A) and CD8+
(FIG. 11B) CAR+ T cells present in the tumor at 14 days after
administration of cells expressing anti-ROR1 CAR-F, CAR-A or the
R12 reference CAR.
[0135] FIGS. 12A-12C depict bioluminescence images assessed up to
approximately day 49 post administration of a low dose (FIG. 12B)
or a high dose (FIG. 12C) of cells expressing anti-ROR1 CAR-F,
CAR-A or the R12 reference CAR in MDA-MB-231 triple negative breast
cancer mouse model. As a control, mice were administered cells not
expressing a CAR (mock) or were untreated (FIG. 12A).
[0136] FIGS. 13A-13C depict the mean measured total flux (p/s) from
the bioluminescence imaging, assessed up to approximately day 49
post administration of a low dose (FIG. 13A) or a high dose (FIG.
13B) of cells expressing anti-ROR1 CAR-F, CAR-A or the R12
reference CAR in MDA-MB-231 triple negative breast cancer mouse
model, shown as group means.+-.standard error. FIG. 13C depicts the
measured total flux (p/s) of individual mice. As a control, mice
were administered cells not expressing a CAR (mock) or were
untreated.
[0137] FIGS. 14A-14E depict the changes in the mean and individual
tumor volume in MDA-MB-231 triple negative breast cancer model mice
administered cells expressing anti-ROR1 CAR-F, CAR-A or the R12
reference CAR, at the low dose (mean: FIG. 14A) or the high dose
(mean: FIG. 14B). The changes in the mean tumor volume following
administration of T cells expressinganti-ROR1 CAR F and reference
CAR R12, up to a further time point in the same study are depicted
for the high dose (FIG. 14C) and low dose (FIG. 14D). Results of
tumor volume for individual treated mice at the high dose or low
dose for each treated condition are shown in FIG. 14E. Mean tumor
volume is depicted as group means.+-.standard error up to the last
day that all mice in treatment groups survived. As a control, mice
were administered cells not expressing a CAR (mock) or were
untreated.
[0138] FIGS. 15A and 15B depict the Kaplan-Meier survival curve in
MDA-MB-231 triple negative breast cancer model mice administered
cells expressing anti-ROR1 CAR-F or the R12 reference CAR, at the
low dose (FIG. 15B) or the high dose (FIG. 15A). As a control, mice
were administered cells not expressing a CAR (mock) or were
untreated.
[0139] FIGS. 16A-16D show the average number of CD4+ (FIGS. 16A and
16C) and CD8+ (FIGS. 16B and 16D) CAR-expressing cells in the blood
of the animal were determined at days 7, 14, 21 and 30 after
administration after administration of cells expressing anti-ROR1
CAR-F, CAR-A or the R12 reference CAR, at the low dose (FIGS. 16A
and 16B) or a high dose (FIGS. 16C and 16D). The average number of
CD3+ CAR-expressing cells (CD45+ CD3+ CAR+) in the blood of each
mouse administered cells expressing anti-ROR1 CAR-F or the R12
reference CAR also was determined at days 7, 14, 21 and 30 are
shown in FIG. 16E. As shown, cells expressing anti-ROR1 CAR-F
exhibited high expansion in the MDA-MB-231 mouse model, when
administered at both high and low doses.
[0140] FIG. 17 depicts the loss of Rapid Red labeled target cells
(CD4/CD8 T cell depleted leukapheresis samples) co-cultured for
approximately 6 days with engineered cells expressing anti-ROR1
CAR-F, anti-CD19 CAR or the R12 reference CAR generated from with
from two primary CLL donors, at two E:T ratios.
[0141] FIG. 18A shows the results of a cytotoxicity assay by flow
cytometry, showing the loss of CD19+ target cells or CD19+ ROR1+
target cells (CD4/CD8 T cell depleted leukapheresis samples)
co-cultured for approximately 4 days with engineered cells
expressing anti-ROR1 CAR-F, anti-CD19 CAR or the R12 reference CAR
generated from two primary CLL donors, at two E:T ratios. FIG. 18B
depicts the proliferation of CAR-expressing cells (anti-ROR1 CAR-F,
anti-CD19 CAR or the R12 reference CAR) labeled with CellTrace.TM.
Violet (CTV) cell proliferation reagent, and co-cultured with
target cells from the subjects with CLL at an E:T ratio of 2.5:1
and 0.25:1. FIG. 18C shows the production of IFN-.gamma.,
TNF-.alpha. and IL-2, as assessed from the supernatant of the
co-culture on day 4. Mock treated cells or target cells only were
compared as controls.
[0142] FIGS. 19A-19D depict the changes in the mean and individual
tumor volume in a mouse model of mantel cell lymphoma (MCL),
implanted with firefly luciferase and green fluorescent protein
(FfLuc-GFP)-expressing human mantel cell lymphoma (MCL) JeKo-1
cells administered cells expressing anti-ROR1 CAR-F, anti-CD19 or
the R12 reference CAR, at the high dose (mean: FIG. 19A;
individual: FIG. 19C) or the low dose (mean: FIG. 19B; individual:
FIG. 19D) or. As a control, mice were administered cells not
expressing a CAR (mock) or were untreated.
[0143] FIGS. 20A-20B depict the Kaplan-Meier survival curve in a
mouse model of MCL administered cells expressing anti-ROR1 CAR-F,
anti-CD19 or the R12 reference CAR, at the high dose (FIG. 20A) or
the low dose (FIG. 20B). As a control, mice were administered cells
not expressing a CAR (mock) or were untreated.
[0144] FIG. 21 shows the average number of average number of CD3+
CAR-expressing cells in the blood of a mouse model of MCL,
determined at days 7, 14, 21 and 28 after administration of
engineered cells expressing anti-ROR1 CAR-F, anti-CD19 or the R12
reference CAR, at the low dose or a high dose.
[0145] FIGS. 22A-22E depict binding of various recombinantly
produced anti-ROR1 scFv-mFcs, including anti-ROR1 scFv ROR1-1 (SEQ
ID NO: 118; FIG. 22A), ROR1-2 (SEQ ID NO: 127; FIG. 22B), ROR1-3
(SEQ ID NO: 109; FIG. 22C) and ROR1-4 (SEQ ID NO: 134; FIG. 22D),
the scFv binding domains of CAR-A, CAR-F, CAR-G, CAR-I, CAR-R and
CAR-B1, and the scFv antigen binding domain of the reference CAR
R12 (SEQ ID NO: 142; FIG. 22E), to with a C-terminal 6.times.His
tag (ROR1 ECD 6.times.His), assessed by surface plasmon resonance
(SPR) using multi-cycle kinetics implemented by subsequent
injections of recombinant human ROR1 at concentrations of 183,
61.0, 20.33, 6.78, and 2.26 nM.
[0146] FIG. 23 depicts ROR1 expression in response to a 2-fold
increasing dilution series of doxycycline from 512 ng/mL to 0 ng/mL
in a K562-ROR1-TetOn cell line. The ROR1- parental K562 cells, two
endogenously expressing ROR1+ cell lines, the MCL cell line JeKo-1
and the TNBC cell line MDA-MB-231 were used as controls.
[0147] FIG. 24 depicts a dose response curve for production of
IFN.gamma., IL-2 and TNF.alpha. assessed from supernatants of a 72
hour co-culture of engineered cells expressing anti-ROR1 CAR-F or
the reference CAR R12, with K562-ROR1-TetOn at a 4:1 E:T ratio with
the addition of increasing concentrations of doxycycline. Cells not
expressing a CAR (mock) were used as a control.
[0148] FIGS. 25A-25B depict results of a cytotoxicity assay, as
determined by loss of red fluorescent signal from NucLight
Red-labeled K562-ROR1-TetOn cells cultured in the presence of
various concentrations (2 ng/mL-512 ng/mL) of doxycycline and of
engineered cells expressing anti-ROR1 CAR-F or the reference CAR
R12, at a 4:1 E:T ratio, for approximately 72 hours. DMSO, no
doxycycline and untreated cells were used as controls.
[0149] FIG. 26 shows assessment of ROR1 species cross reactivity in
Jurkat Nur77 reporter cells expressing either the ROR1 CAR-F or the
ROR1 R12 CAR co-cultured with CT26 cells engineered to express
human ROR1 (hROR1) or murine ROR1 (mROR1). K562 cells and
unmodified CT26 cells were used as non-specific controls.
DETAILED DESCRIPTION
[0150] Provided are receptor tyrosine kinase-like orphan receptor 1
(ROR1)-binding molecules, such as antibodies (including
antigen-binding antibody fragments, such as single chain fragments,
including single chain Fv fragments (scFvs)) and recombinant
receptors, including chimeric receptors containing such antibodies
or fragments and nucleic acids encoding such antibodies, fragments
or recombinant receptors. In some aspects, provided are antibodies,
fragments and chimeric antigen receptors (CARs) targeting or
directed to ROR1 and ROR1-expressing cells and disease. It is
observed that ROR1 is expressed in cells or tissues associated with
certain diseases and conditions such as malignancies, e.g., on
malignant plasma cells such as from relapsed or newly diagnosed
myeloma patients, for example, with little expression on normal
tissues. Among the provided embodiments are approaches useful in
the treatment of diseases and conditions and/or for targeting such
cell types, including nucleic acid molecules that encode
ROR1-binding antibodies, fragments or receptors and the encoded
antibodies or antigen-binding fragments and receptors. Also
provided are compositions and articles of manufacture comprising
the same. The receptors generally can contain antibodies (including
antigen-binding antibody fragments, such as heavy chain variable
(V.sub.H) regions, single domain antibody fragments and single
chain fragments, including scFvs) specific for ROR1, for example as
the antigen-binding domain. Also provided are cells, such as
engineered or recombinant cells, expressing such ROR1-binding
receptors, e.g., anti-ROR1 CARs and/or containing nucleic acids
encoding such receptors, and compositions and articles of
manufacture and therapeutic doses containing such cells. Also
provided are methods of making and using the antibodies and
fragments as well as cells expressing or containing the antibodies
and fragments, such as for production of the antibodies or
fragments thereof. Also provided are compositions, including
pharmaceutical compositions, containing such antibodies,
antigen-binding fragments, receptors or cells, and conjugates
comprising such antibodies or fragments. In some aspects, the
provided compositions, antibodies, antigen-binding fragments,
receptors or cells can be used in connection with a therapy or a
method of treatment.
[0151] Therapies targeting ROR1, such as with anti-ROR1 antibodies
or adoptive cell therapies (including those involving the
administration of cells expressing chimeric receptors such as
chimeric antigen receptors (CARs) and/or other recombinant antigen
receptors, specific for ROR1, as well as other adoptive immune cell
and adoptive T cell therapies) can be effective in the treatment of
cancer and other diseases and disorders, for example, ROR1. In
certain contexts, available approaches to adoptive cell therapy may
not always be entirely satisfactory. In some aspects, the ability
of the administered cells to recognize and bind to a target, e.g.,
target antigen such as ROR1, to traffic, localize to and
successfully enter appropriate sites within the subject, tumors,
and environments thereof, to become activated, expand, to exert
various effector functions, including cytotoxic killing and
secretion of various factors such as cytokines, to persist,
including long-term, to differentiate, transition or engage in
reprogramming into certain phenotypic states to provide effective
and robust recall responses following clearance and re-exposure to
target ligand or antigen, and avoid or reduce exhaustion, anergy,
terminal differentiation, and/or differentiation into a suppressive
state.
[0152] In some contexts, properties of particular target antigens
that the antibodies or recombinant receptors containing
antigen-binding domains specifically bind, recognize or target, can
that affect the activity of the receptor. In some contexts, ROR1 is
expressed by certain cancers and is an attractive therapeutic
target for cell therapy. Improved strategies are needed for optimal
responses to antibody or cell therapies, in particular, for
recombinant receptors that specifically bind, recognize or target
ROR1. Provided are embodiments that meet such needs.
[0153] In some aspects, the provided embodiments are based on
observations that administration of engineered cells expressing the
provided ROR1 binding molecules, such as chimeric antigen receptors
(CARs), exhibit an improved antigen-specific activity, signaling
and function, high anti-tumor activity, consistent
antigen-dependent activity or signaling, greater or prolonged in
vivo expansion, and improved persistence when administered, while
exhibiting minimal antigen-independent activity or signaling or
cross-reactivity to a different antigen. Such antibodies and
recombinant receptors can be used to facilitate safe and effective
treatment of particular diseases and disorders, such as those
associated with expression of ROR1.
[0154] In some contexts, optimal response to therapy can depend on
the ability of the antibody or antigen-binding fragment, or
recombinant receptors that contain such antibody or antigen-binding
fragment as antigen-binding domains, to recognize the target
antigen. In some aspects, it is observed herein that the binding
affinity, specificity or kinetics of binding to ROR1 of certain
provided antibody or antigen-binding fragments, such as when
present as the antigen-binding domain of a recombinant receptor
(e.g. CAR), can be associated with an improved or greater response
to the therapy. In some aspects, the provided embodiments are based
on observations that provided binding molecules exhibit a lower
binding affinity and/or a faster dissociation rate constant
(k.sub.off or k.sub.d; fast off-rate) to ROR1 compared to available
antibodies, and also exhibit improved anti-tumor activity, greater
or prolonged in vivo expansion, improved persistence, and/or
reduced antigen-independent activity or signaling. In some aspects,
engineered cells (e.g. T cells) expressing a recombinant receptor
containing an antigen-binding domain (e.g., antibody or
antigen-binding fragment thereof) with a lower binding affinity
(e.g. higher equilibrium dissociation constant) may exhibit
substantially improved in vivo expansion, increased persistence,
greater or improved antigen-specific anti-tumor activity and
prolonged survival, including against various different types of
tumors.
[0155] In some aspects, engineered cells (e.g. T cells) expressing
a recombinant receptor containing an antigen-binding domain (e.g.,
antibody or antigen-binding fragment thereof) with a faster
dissociation rate constant (k.sub.off or k.sub.d; fast off-rate)
may exhibit substantially improved in vivo expansion, increased
persistence, greater or improved antigen-specific anti-tumor
activity and prolonged survival, including against various
different types of tumors. In some aspects, engineered cells (e.g.
T cells) expressing a recombinant receptor containing an
antigen-binding domain (e.g., antibody or antigen-binding fragment
thereof) with a lower binding affinity (e.g. higher equilibrium
dissociation constant) and a faster dissociation rate constant
(k.sub.off or k.sub.d; fast off-rate) may exhibit substantially
improved in vivo expansion, increased persistence, greater or
improved antigen-specific anti-tumor activity and prolonged
survival, including against various different types of tumors.
Without wishing to be bound by theory, in some aspects, it is
observed herein that the increased antigen-binding off-rate of the
binding domain of an exemplary anti-ROR1 CAR for ROR1 binding
potentially contributes to increased sensitivity of the CAR to low
antigen levels.
[0156] In some contexts, optimal response to therapy such as cell
therapy can depend on the ability of the engineered recombinant
receptors such as CARs, to be consistently and reliably expressed
on the surface of the cells and/or bind the target antigen. For
example, in some cases, heterogeneity of the transcribed RNA from
an introduced transgene (e.g., encoding the recombinant receptor)
can affect the expression and/or activity of the recombinant
receptor, in some cases when expressed in a cell, such as a human T
cell, used in cell therapy.
[0157] In some contexts, the length and type of spacer in the
recombinant receptor, such as a CAR, can affect the expression,
activity and/or function of the receptor.
[0158] Also, in some contexts, certain recombinant receptors can
exhibit antigen-independent activity or signaling (also known as
"tonic signaling"), which could lead to undesirable effects, such
as due to increased differentiation and/or exhaustion of T cells
that express the recombinant receptor. In some aspects, such
activities may limit the T cell's activity, effect or potency. In
some cases, during engineering and ex vivo expansion of the cells
for recombinant receptor expression, the cells may exhibit
phenotypes indicative of exhaustion, due to tonic signaling through
the recombinant receptor.
[0159] All publications, including patent documents, scientific
articles and databases, referred to in this application are
incorporated by reference in their entirety for all purposes to the
same extent as if each individual publication were individually
incorporated by reference. If a definition set forth herein is
contrary to or otherwise inconsistent with a definition set forth
in the patents, applications, published applications and other
publications that are herein incorporated by reference, the
definition set forth herein prevails over the definition that is
incorporated herein by reference.
[0160] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
I. ROR1-BINDING MOLECULES
[0161] Provided in some aspects are ROR1-binding molecules, such as
ROR1-binding polypeptides. Such binding molecules include
antibodies (including antigen-binding fragments) that specifically
bind to ROR1 proteins, such as a human ROR1 protein. Also among the
binding molecules are polypeptides containing such antibodies,
including single chain cell surface proteins, e.g., recombinant
receptors such as chimeric antigen receptors (CARs), containing
such antibodies. Provided in some aspects are ROR1-binding cell
surface proteins, such as recombinant receptors or chimeric antigen
receptors (CARs) that bind ROR1 molecules and polynucleotides
encoding ROR1 binding cell surface proteins, such as recombinant
receptors (e.g., CARs), and cells expressing such receptors. Also
provided are polynucleotides containing nucleic acids sequences
encoding all or a portion of such antibodies, antigen-binding
fragments and binding molecules, such as those described in Section
I.A or I.D. Exemplary of such polynucleotides include those
described in Section I.E. In some aspects the polynucleotides can
be introduced into a cell to generate an engineered cell that
contains or expresses the provided binding molecules, e.g.,
ROR1-binding antibodies, antigen-binding domains and receptors,
such as CARs.
[0162] A. ROR1-Targeting Antibodies
[0163] Provided are anti-ROR1 polypeptides, including antibodies
and functional antigen-binding fragments. Among the ROR1-binding
polypeptides are antibodies, such as single-chain antibodies (e.g.,
antigen binding antibody fragments), such as those containing a
heavy chain variable (V.sub.H) region and/or a light chain variable
(V.sub.L) region, or a portion thereof. In some embodiments, the
antibodies or antigen-binding fragments include a V.sub.H and a
V.sub.L, such as single chain Fv fragments (scFvs). The antibodies
include antibodies that specifically bind to ROR1, e.g., human
ROR1. Among the provided anti-ROR1 antibodies are human antibodies,
or antibodies that are modified from or variant of human
antibodies. The antibodies include isolated antibodies. Also
provided are ROR1-binding molecules containing such antibodies,
such as single-chain proteins, fusion proteins, conjugates and/or
recombinant receptors such as chimeric receptors, including antigen
receptors. In some aspects, the ROR1-binding molecules include
isolated molecules.
[0164] Also provided are ROR1-binding cell surface proteins, such
as ROR1-binding recombinant receptors. The ROR1-binding cell
surface proteins can contain the provided antibodies (e.g.,
antigen-binding antibody fragments) that specifically bind to ROR1,
such as to ROR1 proteins, such as human ROR1 protein. In some
aspects, the provided binding molecules bind to an extracellular
portion of ROR1. In some examples, the recombinant receptors are
chimeric antigen receptors, such as those containing anti-ROR1
antibodies or antigen-binding fragments thereof.
[0165] Also provided are polynucleotides containing nucleic acids
sequences encoding all or a portion of such antibodies,
antigen-binding fragments and binding molecules. The provided
polynucleotides can be incorporated into constructs, such as
deoxyribonucleic acid (DNA) or RNA constructs, such as those that
can be introduced into cells for expression of the encoded
ROR1-binding antibodies, antigen-binding fragments, conjugates or
receptors, e.g., anti-ROR1 CARs. In some aspects, the encoded
antibodies, antigen-binding fragments, conjugates and receptors,
such as those containing ROR1-binding polypeptides, and
compositions and articles of manufacture and uses of the same, also
are provided.
[0166] The term "antibody" herein is used in the broadest sense and
includes polyclonal and monoclonal antibodies, including intact
antibodies and functional (antigen-binding) antibody fragments,
including fragment antigen binding (Fab) fragments, F(ab').sub.2
fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG)
fragments, heavy chain variable (V.sub.H) regions capable of
specifically binding the antigen, single chain antibody fragments,
including single chain variable fragments (scFv), and single domain
antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term
encompasses genetically engineered and/or otherwise modified forms
of immunoglobulins, such as intrabodies, peptibodies, chimeric
antibodies, fully human antibodies, humanized antibodies, and
heteroconjugate antibodies, multispecific, e.g., bispecific or
trispecific, antibodies, diabodies, triabodies, and tetrabodies,
tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term
"antibody" should be understood to encompass functional antibody
fragments thereof also referred to herein as "antigen-binding
fragments." The term also encompasses intact or full-length
antibodies, including antibodies of any class or sub-class,
including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
[0167] The terms "complementarity determining region," and "CDR,"
synonymous with "hypervariable region" or "HVR," are known to refer
to non-contiguous sequences of amino acids within antibody variable
regions, which confer antigen specificity and/or binding affinity.
In general, there are three CDRs in each heavy chain variable
region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain
variable region (CDR-L1, CDR-L2, CDR-L3). "Framework regions" and
"FR" are known to refer to the non-CDR portions of the variable
regions of the heavy and light chains. In general, there are four
FRs in each full-length heavy chain variable region (FR-H1, FR-H2,
FR-H3, and FR-H4), and four FRs in each full-length light chain
variable region (FR-L1, FR-L2, FR-L3, and FR-L4).
[0168] The precise amino acid sequence boundaries of a given CDR or
FR can be readily determined using any of a number of well-known
schemes, including those described by Kabat et al. (1991),
"Sequences of Proteins of Immunological Interest," 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,
927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol.
Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact
analysis and binding site topography," J. Mol. Biol. 262, 732-745."
("Contact" numbering scheme); Lefranc M P et al., "IMGT unique
numbering for immunoglobulin and T cell receptor variable domains
and Ig superfamily V-like domains," Dev Comp Immunol, 2003 January;
27(1):55-77 ("IMGT" numbering scheme); Honegger A and Pluckthun A,
"Yet another numbering scheme for immunoglobulin variable domains:
an automatic modeling and analysis tool," J Mol Biol, 2001 Jun. 8;
309(3):657-70, ("Aho" numbering scheme); Martin et al., "Modeling
antibody hypervariable loops: a combined algorithm," PNAS, 1989,
86(23):9268-9272, ("AbM" numbering scheme); and Ye et al.,
"IgBLAST: an immunoglobulin variable domain sequence analysis
tool," Nucleic Acids Res. 2013 July; 41 (Web Server issue):W34-40,
("IgBLAST numbering scheme).
[0169] The boundaries of a given CDR or FR may vary depending on
the scheme used for identification. For example, the Kabat scheme
is based on structural alignments, while the Chothia scheme is
based on structural information. Numbering for both the Kabat and
Chothia schemes is based upon the most common antibody region
sequence lengths, with insertions accommodated by insertion
letters, for example, "30a," and deletions appearing in some
antibodies. The two schemes place certain insertions and deletions
("indels") at different positions, resulting in differential
numbering. The Contact scheme is based on analysis of complex
crystal structures and is similar in many respects to the Chothia
numbering scheme. The AbM scheme is a compromise between Kabat and
Chothia definitions based on that used by Oxford Molecular's AbM
antibody modeling software. The IgBLAST scheme is based on matching
to germline V, D and J genes, and can be determined using National
Center for Biotechnology Information (NCBI)'s IgBLAST tool.
[0170] Table 1, below, lists exemplary position boundaries of
CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by
Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1,
residue numbering is listed using both the Kabat and Chothia
numbering schemes. FRs are located between CDRs, for example, with
FR-L1 located before CDR-L1, FR-L2 located between CDR-L1 and
CDR-L2, FR-L3 located between CDR-L2 and CDR-L3 and so forth. It is
noted that because the shown Kabat numbering scheme places
insertions at H35A and H35B, the end of the Chothia CDR-H1 loop
when numbered using the shown Kabat numbering convention varies
between H32 and H34, depending on the length of the loop.
TABLE-US-00001 TABLE 1 Boundaries of CDRs according to various
numbering schemes. CDR Kabat Chothia AbM Contact CDR-L1 L24--L34
L24--L34 L24--L34 L30--L36 CDR-L2 L50--L56 L50--L56 L50--L56
L46--L55 CDR-L3 L89--L97 L89--L97 L89--L97 L89--L96 CDR-H1
H31--H35B H26--H32.34 H26--H35B H30--H35B (Kabat Numbering.sup.1)
CDR-H1 H31--H35 H26--H32 H26--H35 H30--H35 (Chothia
Numbering.sup.2) CDR-H2 H50--H65 H52--H56 H50--H58 H47--H58 CDR-H3
H95--H102 H95--H102 H95--H102 H93--H101 .sup.1Kabat et al. (1991),
"Sequences of Proteins of Immunological Interest," 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, MD
.sup.2Al-Lazikani et al., (1997) JMB 273, 927-948
[0171] Thus, unless otherwise specified, a "CDR" or "complementary
determining region," or individual specified CDRs (e.g., CDR-H1,
CDR-H2, CDR-H3), of a given antibody or region thereof, such as a
variable region thereof, should be understood to encompass a (or
the specific) complementary determining region as defined by any of
the aforementioned schemes, or other known schemes. For example,
where it is stated that a particular CDR (e.g., a CDR-H3) contains
the amino acid sequence of a corresponding CDR in a given V.sub.H
or V.sub.L region amino acid sequence, it is understood that such a
CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within
the variable region, as defined by any of the aforementioned
schemes, or other known schemes. In some embodiments, specific CDR
sequences are specified. Exemplary CDR sequences of provided
antibodies are described using various numbering schemes (see e.g.
Table 2), although it is understood that a provided antibody can
include CDRs as described according to any of the other
aforementioned numbering schemes or other known numbering
schemes.
[0172] Likewise, unless otherwise specified, a FR or individual
specified FR(s) (e.g., FR-H1, FR-H2, FR-H3, FR-H4), of a given
antibody or region thereof, such as a variable region thereof,
should be understood to encompass a (or the specific) framework
region as defined by any of the known schemes. In some instances,
the scheme for identification of a particular CDR, FR, or FRs or
CDRs is specified, such as the CDR as defined by the Kabat,
Chothia, AbM, IgBLAST, IMGT, or Contact method, or other known
schemes. In other cases, the particular amino acid sequence of a
CDR or FR is given.
[0173] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable regions of the heavy
chain and light chain (V.sub.H and V.sub.L, respectively) of a
native antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three CDRs.
(See, e.g., Kindt et al. Kuby Immunology, 6th ed., W. H. Freeman
and Co., page 91 (2007). A single V.sub.H or V.sub.L domain may be
sufficient to confer antigen-binding specificity. Furthermore,
antibodies that bind a particular antigen may be isolated using a
V.sub.H or V.sub.L domain from an antibody that binds the antigen
to screen a library of complementary V.sub.L or V.sub.H domains,
respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887
(1993); Clarkson et al., Nature 352:624-628 (1991).
[0174] Among the provided antibodies are antibody fragments. An
"antibody fragment" or "antigen-binding fragment" refers to a
molecule other than an intact antibody that comprises a portion of
an intact antibody that binds the antigen to which the intact
antibody binds. Examples of antibody fragments include but are not
limited to Fv, Fab, Fab', Fab'-SH, F(ab').sub.2; diabodies; linear
antibodies; heavy chain variable (V.sub.H) regions, single-chain
antibody molecules such as scFvs and single-domain antibodies
comprising only the V.sub.H region; and multispecific antibodies
formed from antibody fragments. In some embodiments, the antibody
is or comprises an antibody fragment comprising a variable heavy
chain (V.sub.H) and a variable light chain (V.sub.L) region. In
particular embodiments, the antibodies are single-chain antibody
fragments comprising a heavy chain variable (V.sub.H) region and/or
a light chain variable (V.sub.L) region, such as scFvs.
[0175] Single-domain antibodies (sdAbs) are antibody fragments
comprising all or a portion of the heavy chain variable region or
all or a portion of the light chain variable region of an antibody.
In certain embodiments, a single-domain antibody is a human
single-domain antibody.
[0176] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells. In some
embodiments, the antibodies are recombinantly-produced fragments,
such as fragments comprising arrangements that do not occur
naturally, such as those with two or more antibody regions or
chains joined by synthetic linkers, e.g., peptide linkers, and/or
that are may not be produced by enzyme digestion of a
naturally-occurring intact antibody. In some aspects, the antibody
fragments are scFvs.
[0177] A "humanized" antibody is an antibody in which all or
substantially all CDR amino acid residues are derived from
non-human CDRs and all or substantially all FR amino acid residues
are derived from human FRs. A humanized antibody optionally may
include at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of a non-human antibody,
refers to a variant of the non-human antibody that has undergone
humanization, typically to reduce immunogenicity to humans, while
retaining the specificity and affinity of the parental non-human
antibody. In some embodiments, some FR residues in a humanized
antibody are substituted with corresponding residues from a
non-human antibody (e.g., the antibody from which the CDR residues
are derived), e.g., to restore or improve antibody specificity or
affinity.
[0178] Among the provided anti-ROR1 antibodies are human
antibodies. A "human antibody" is an antibody with an amino acid
sequence corresponding to that of an antibody produced by a human
or a human cell, or non-human source that utilizes human antibody
repertoires or other human antibody-encoding sequences, including
human antibody libraries. The term excludes humanized forms of
non-human antibodies comprising non-human antigen-binding regions,
such as those in which all or substantially all CDRs are non-human.
The term includes antigen-binding fragments of human
antibodies.
[0179] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic animals, the endogenous
immunoglobulin loci have generally been inactivated. Human
antibodies also may be derived from human antibody libraries,
including phage display and cell-free libraries, containing
antibody-encoding sequences derived from a human repertoire.
[0180] Among the provided antibodies are monoclonal antibodies,
including monoclonal antibody fragments. The term "monoclonal
antibody" as used herein refers to an antibody obtained from or
within a population of substantially homogeneous antibodies, i.e.,
the individual antibodies comprising the population are identical,
except for possible variants containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
epitopes, each monoclonal antibody of a monoclonal antibody
preparation is directed against a single epitope on an antigen. The
term is not to be construed as requiring production of the antibody
by any particular method. A monoclonal antibody may be made by a
variety of techniques, including but not limited to generation from
a hybridoma, recombinant DNA methods, phage-display and other
antibody display methods.
[0181] The terms "polypeptide" and "protein" are used
interchangeably to refer to a polymer of amino acid residues, and
are not limited to a minimum length. Polypeptides, including the
provided antibodies and antibody chains and other peptides, e.g.,
linkers and ROR1-binding peptides, may include amino acid residues
including natural and/or non-natural amino acid residues. The terms
also include post-expression modifications of the polypeptide, for
example, glycosylation, sialylation, acetylation, phosphorylation,
and the like. In some aspects, the polypeptides may contain
modifications with respect to a native or natural sequence, as long
as the protein maintains the desired activity. These modifications
may be deliberate, as through site-directed mutagenesis, or may be
accidental, such as through mutations of hosts which produce the
proteins or errors due to PCR amplification.
[0182] 1. Exemplary Antibodies
[0183] In some embodiments, the antibody, e.g., the anti-ROR1
antibody, e.g., antigen-binding antibody fragment, contains a heavy
and/or light chain variable (V.sub.H or V.sub.L) region sequence as
described, or a sufficient antigen-binding portion thereof. In some
embodiments, the antibody, e.g., the anti-ROR1 antibody, e.g.,
antigen-binding antibody fragment, contains a heavy chain variable
region (V.sub.H) sequence and/or a light chain variable region
(V.sub.L) sequence as described, or a sufficient antigen-binding
portion thereof. In some embodiments, the antibody, e.g., the
anti-ROR1 antibody, e.g., antigen-binding antibody fragment, is a
single chain fragment, such as a single chain Fv (scFv) fragment.
In some aspects, the scFv comprises a V.sub.H region and a V.sub.L
region. In some embodiments, the antibody, e.g., the anti-ROR1
antibody, e.g., antigen-binding antibody fragment, is a single
domain antibody (sdAb), such as an antibody that contains a V.sub.H
region only.
[0184] In some embodiments, the anti-ROR1 antibody, e.g.,
antigen-binding antibody fragment, contains a V.sub.H region
sequence or sufficient antigen-binding portion thereof that
contains a heavy chain complementarity determining region 1
(CDR-H1), a heavy chain complementarity determining region 2
(CDR-H2) and/or a heavy chain complementarity determining region 3
(CDR-H3) as described. In some embodiments, the anti-ROR1 antibody,
e.g., antigen-binding antibody fragment, contains a V.sub.H region
sequence or sufficient antigen-binding portion thereof that
contains a CDR-H1, a CDR-H2 and a CDR-H3 as described. In some
embodiments, the anti-ROR1 antibody, e.g., antigen-binding antibody
fragment, contains a V.sub.L region sequence or sufficient
antigen-binding portion that contains a light chain complementarity
determining region 1 (CDR-L1), a light chain complementarity
determining region 2 (CDR-L2), and/or a light chain complementarity
determining region 3 (CDR-L3) as described. In some embodiments,
the anti-ROR1 antibody, e.g., antigen-binding antibody fragment,
contains a V.sub.L region sequence or sufficient antigen-binding
portion that contains a CDR-L1, a CDR-L2 and a CDR-L3 as
described.
[0185] In some embodiments, the anti-ROR1 antibody, e.g.,
antigen-binding antibody fragment, contains a V.sub.H region
sequence that contains a CDR-H1, a CDR-H2 and/or a CDR-H3 as
described and contains a V.sub.L region sequence that contains a
CDR-L1, a CDR-L2 and/or a CDR-L3 as described. In some embodiments,
the anti-ROR1 antibody, e.g., antigen-binding antibody fragment,
contains a V.sub.H region sequence that contains a CDR-H1, a CDR-H2
and a CDR-H3 as described and contains a V.sub.L region sequence
that contains a CDR-L1, a CDR-L2 and a CDR-L3 as described. Also
among the provided antibodies and fragment thereof are those having
sequences at least at or about 85%, at or about 86%, at or about
87%, at or about 88%, at or about 89%, at or about 90%, at or about
91%, at or about 92%, at or about 93%, at or about 94%, at or about
95%, at or about 96%, at or about 97%, at or about 98%, or at or
about 99% identical to such a sequence, e.g., any of the CDR-H1,
CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, V.sub.H, V.sub.L, scFv
sequences or other sequences of the antibodies of fragment thereof
described herein. In some aspects, among the provided antibodies
and fragment thereof are those having sequences at least at or
about 85% sequence identity to any such sequences. In some aspects,
among the provided antibodies and fragment thereof are those having
sequences at least at or about 86% sequence identity to any such
sequences. In some aspects, among the provided antibodies and
fragment thereof are those having sequences at least at or about
87% sequence identity to any such sequences. In some aspects, among
the provided antibodies and fragment thereof are those having
sequences at least at or about 88% sequence identity to any such
sequences. In some aspects, among the provided antibodies and
fragment thereof are those having sequences at least at or about
89% sequence identity to any such sequences. In some aspects, among
the provided antibodies and fragment thereof are those having
sequences at least at or about 90% sequence identity to any such
sequences. In some aspects, among the provided antibodies and
fragment thereof are those having sequences at least at or about
91% sequence identity to any such sequences. In some aspects, among
the provided antibodies and fragment thereof are those having
sequences at least at or about 92% sequence identity to any such
sequences. In some aspects, among the provided antibodies and
fragment thereof are those having sequences at least at or about
93% sequence identity to any such sequences. In some aspects, among
the provided antibodies and fragment thereof are those having
sequences at least at or about 94% sequence identity to any such
sequences. In some aspects, among the provided antibodies and
fragment thereof are those having sequences at least at or about
95% sequence identity to any such sequences. In some aspects, among
the provided antibodies and fragment thereof are those having
sequences at least at or about 96% sequence identity to any such
sequences. In some aspects, among the provided antibodies and
fragment thereof are those having sequences at least at or about
97% sequence identity to any such sequences. In some aspects, among
the provided antibodies and fragment thereof are those having
sequences at least at or about 98% sequence identity to any such
sequences. In some aspects, among the provided antibodies and
fragment thereof are those having sequences at least at or about
99% sequence identity to any such sequences.
[0186] In some embodiments, the antibody is an sdAb comprising only
a V.sub.H region sequence or a sufficient antigen-binding portion
thereof, such as any of the V.sub.H sequences described herein
(e.g., a CDR-H1, a CDR-H2, a CDR-H3 and/or a CDR-H4). In some
embodiments, the antibodies or antigen-binding fragments include
those that are single domain antibodies, containing a V.sub.H
region that, without pairing with a V.sub.L region) and/or without
any additional antibody domain or binding site, are capable of
specifically binding to ROR1.
[0187] In some embodiments, the V.sub.H region of an antibody or
antigen-binding fragment thereof comprises a CDR-H1, a CDR-H2
and/or a CDR-H3 according to Kabat numbering. In some embodiments,
the V.sub.H region of an antibody or antigen-binding fragment
thereof comprises a CDR-H1, a CDR-H2 and/or a CDR-H3 according to
Chothia numbering. In some embodiments, the V.sub.H region of an
antibody or antigen-binding fragment thereof comprises a CDR-H1, a
CDR-H2 and/or a CDR-H3 according to AbM numbering. In some
embodiments, the V.sub.H region of an antibody or antigen-binding
fragment thereof comprises a CDR-H1, a CDR-H2 and/or a CDR-H3
according to IgBLAST numbering. In some embodiments, the V.sub.H
region of an antibody or antigen-binding fragment thereof comprises
a CDR-H1, a CDR-H2 and/or a CDR-H3 according to Kabat, Chothia,
AbM, IMGT or IgBLAST numbering, or other numbering schemes.
[0188] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, has a heavy chain variable (V.sub.H) region having
the amino acid sequence set forth in SEQ ID NO: 112, 121, 103 or
130, or an amino acid sequence that has at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% sequence identity to the V.sub.H region amino acid sequence set
forth in SEQ ID NO: 112, 121, 103 or 130, or contains a heavy chain
complementarity determining region 1 (CDR-H1), a heavy chain
complementarity determining region 2 (CDR-H2) and/or a heavy chain
complementarity determining region 3 (CDR-H3) present in such a
V.sub.H sequence, such as one that contains a CDR-H1, a CDR-H2, and
a CDR-H3 present in such a V.sub.H sequence. In some embodiments of
the antibody, antigen-binding fragment, receptor, e.g., CAR,
conjugates or binding molecules provided herein, has a V.sub.H
region having the amino acid sequence set forth in SEQ ID NO: 112
or 121, or an amino acid sequence that has at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the V.sub.H region amino acid
sequence set forth in SEQ ID NO: 112 or 121, or contains a CDR-H1,
a CDR-H2 and/or a CDR-H3 present in such a V.sub.H sequence, such
as one that contains a CDR-H1, a CDR-H2, and a CDR-H3 present in
such a V.sub.H sequence.
[0189] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, has a V.sub.H region having the amino acid
sequence set forth in SEQ ID NO: 112, or an amino acid sequence
that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
V.sub.H region amino acid sequence set forth in SEQ ID NO: 112, or
contains a CDR-H1, a CDR-H2 and/or a CDR-H3 present in such a
V.sub.H sequence, such as one that contains a CDR-H1, a CDR-H2, and
a CDR-H3 present in such a V.sub.H sequence. In some embodiments,
the antibody, e.g., antigen-binding fragment thereof, has a V.sub.H
region having the amino acid sequence set forth in SEQ ID NO: 121,
or an amino acid sequence that has at least at or about 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the V.sub.H region amino acid sequence set
forth in SEQ ID NO: 121, or contains a CDR-H1, a CDR-H2 and/or a
CDR-H3 present in such a V.sub.H sequence, such as one that
contains a CDR-H1, a CDR-H2, and a CDR-H3 present in such a V.sub.H
sequence. In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, has a V.sub.H region having the amino acid
sequence set forth in SEQ ID NO: 103, or an amino acid sequence
that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
V.sub.H region amino acid sequence set forth in SEQ ID NO: 103, or
contains a CDR-H1, a CDR-H2 and/or a CDR-H3 present in such a
V.sub.H sequence, such as one that contains a CDR-H1, a CDR-H2, and
a CDR-H3 present in such a V.sub.H sequence. In some embodiments,
the antibody, e.g., antigen-binding fragment thereof, has a V.sub.H
region having the amino acid sequence set forth in SEQ ID NO: 130,
or an amino acid sequence that has at least at or about 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the V.sub.H region amino acid sequence set
forth in SEQ ID NO: 130, or contains a CDR-H1, a CDR-H2 and/or a
CDR-H3 present in such a V.sub.H sequence, such as one that
contains a CDR-H1, a CDR-H2, and a CDR-H3 present in such a V.sub.H
sequence.
[0190] In some of any of the provided embodiments, the V.sub.H
region comprises a CDR-H1 comprising the sequence set forth in SEQ
ID NO: 67, 82 or 52. In some of any of the provided embodiments,
the V.sub.H region comprises a CDR-H2 comprising the sequence set
forth in SEQ ID NO: 71, 86, 56 or 97. In some of any of the
provided embodiments, the V.sub.H region comprises a CDR-H3
comprising the sequence set forth in SEQ ID NO: 73, 88, 58 or
99.
[0191] In some of any of the provided embodiments, the V.sub.H
region comprises a CDR-H1 comprising the sequence set forth in SEQ
ID NO: 65, 80 or 50. In some of any of the provided embodiments,
the V.sub.H region comprises a CDR-H2 comprising the sequence set
forth in SEQ ID NO: 69, 84, 54 or 95. In some of any of the
provided embodiments, the V.sub.H region comprises a CDR-H3
comprising the sequence set forth in SEQ ID NO: 73, 88, 58 or
99.
[0192] In some of any of the provided embodiments, the V.sub.H
region comprises a CDR-H1 comprising the sequence set forth in SEQ
ID NO: 66, 81 or 51. In some of any of the provided embodiments,
the V.sub.H region comprises a CDR-H2 comprising the sequence set
forth in SEQ ID NO: 70, 85, 55 or 96. In some of any of the
provided embodiments, the V.sub.H region comprises a CDR-H3
comprising the sequence set forth in SEQ ID NO: 73, 88, 58 or
99.
[0193] In some of any of the provided embodiments, the V.sub.H
region comprises a CDR-H1 comprising the sequence set forth in SEQ
ID NO: 68, 83 or 53. In some of any of the provided embodiments,
the V.sub.H region comprises a CDR-H2 comprising the sequence set
forth in SEQ ID NO: 72, 87, 57 or 98. In some of any of the
provided embodiments, the V.sub.H region comprises a CDR-H3
comprising the sequence set forth in SEQ ID NO: 74, 89, 59 or
100.
[0194] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1 comprising
the sequence set forth in SEQ ID NO: 67, 82 or 52, a CDR-H2
comprising the sequence set forth in SEQ ID NO: 71, 86, 56 or 97,
and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 73,
88, 58 or 99. In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1 comprising
the sequence set forth in SEQ ID NO: 65, 80 or 50, a CDR-H2
comprising the sequence set forth in SEQ ID NO: 69, 84, 54 or 95,
and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 73,
88, 58 or 99. In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1 comprising
the sequence set forth in SEQ ID NO: 66, 81 or 51, a CDR-H2
comprising the sequence set forth in SEQ ID NO: 70, 85, 55 or 96,
and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 73,
88, 58 or 99. In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1 comprising
the sequence set forth in SEQ ID NO: 68, 83 or 53, a CDR-H2
comprising the sequence set forth in SEQ ID NO: 72, 87, 57 or 98,
and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 74,
89, 59 or 100.
[0195] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1 comprising
the sequence set forth in SEQ ID NO: 67 or 82, a CDR-H2 comprising
the sequence set forth in SEQ ID NO: 71 or 86, and a CDR-H3
comprising the sequence set forth in SEQ ID NO: 73 or 88. In some
embodiments of the antibody, antigen-binding fragment, receptor,
e.g., CAR, conjugates or binding molecules provided herein, the
V.sub.H region comprises a CDR-H1 comprising the sequence set forth
in SEQ ID NO: 65 or 80, a CDR-H2 comprising the sequence set forth
in SEQ ID NO: 69 or 84, and a CDR-H3 comprising the sequence set
forth in SEQ ID NO: 73 or 88. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.H region comprises a
CDR-H1 comprising the sequence set forth in SEQ ID NO: 66 or 81, a
CDR-H2 comprising the sequence set forth in SEQ ID NO: 70 or 85,
and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 73 or
88. In some embodiments of the antibody, antigen-binding fragment,
receptor, e.g., CAR, conjugates or binding molecules provided
herein, the V.sub.H region comprises a CDR-H1 comprising the
sequence set forth in SEQ ID NO: 68 or 83, a CDR-H2 comprising the
sequence set forth in SEQ ID NO: 72 or 87, and a CDR-H3 comprising
the sequence set forth in SEQ ID NO: 74 or 89.
[0196] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:67, 71
and 73, respectively; SEQ ID NOS:82, 86 and 88; SEQ ID NOS:52, 56
and 58, respectively; or SEQ ID NOS:52, 97 and 99, respectively. In
some embodiments of the antibody, antigen-binding fragment,
receptor, e.g., CAR, conjugates or binding molecules provided
herein, the V.sub.H region comprises a CDR-H1, a CDR-H2 and a
CDR-H3 comprising the sequence set forth in SEQ ID NOS:65, 69 and
73, respectively; SEQ ID NOS:80, 84 and 88, respectively; SEQ ID
NOS:50, 54 and 58, respectively; or SEQ ID NOS:50, 95 and 99,
respectively. In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:66, 70
and 73, respectively; SEQ ID NOS:81, 85 and 88, respectively; SEQ
ID NOS:51, 55 and 58, respectively; or SEQ ID NOS:51, 96 and 99. In
some embodiments of the antibody, antigen-binding fragment,
receptor, e.g., CAR, conjugates or binding molecules provided
herein, the V.sub.H region comprises a CDR-H1, a CDR-H2 and a
CDR-H3 comprising the sequence set forth in SEQ ID NOS:68, 72 and
74, respectively; SEQ ID NOS:83, 87 and 89, respectively; SEQ ID
NOS:53, 57 and 59, respectively; or SEQ ID NOS:53, 98 and 100,
respectively.
[0197] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:67, 71
and 73, respectively. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.H region comprises a
CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in
SEQ ID NOS:65, 69 and 73, respectively. In some embodiments of the
antibody, antigen-binding fragment, receptor, e.g., CAR, conjugates
or binding molecules provided herein, the V.sub.H region comprises
a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth
in SEQ ID NOS:66, 70 and 73, respectively. In some embodiments of
the antibody, antigen-binding fragment, receptor, e.g., CAR,
conjugates or binding molecules provided herein, the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence
set forth in SEQ ID NOS:68, 72 and 74, respectively.
[0198] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:82, 86
and 88, respectively. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.H region comprises a
CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in
SEQ ID NOS:80, 84 and 88, respectively. In some embodiments of the
antibody, antigen-binding fragment, receptor, e.g., CAR, conjugates
or binding molecules provided herein, the V.sub.H region comprises
a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth
in SEQ ID NOS:81, 85 and 88, respectively. In some embodiments of
the antibody, antigen-binding fragment, receptor, e.g., CAR,
conjugates or binding molecules provided herein, the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence
set forth in SEQ ID NOS:83, 87 and 89, respectively.
[0199] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3, respectively, comprising the amino acid sequence of a
CDR-H1, a CDR-H2 and a CDR-H3 contained within the V.sub.H region
amino acid sequence set forth in any one of SEQ ID NOs: 112, 121,
103 or 130. In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 contained within SEQ ID NO:112 or 121.
[0200] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 contained within SEQ ID NO:112. In some embodiments of
the antibody, antigen-binding fragment, receptor, e.g., CAR,
conjugates or binding molecules provided herein, the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within SEQ ID
NO:121. In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 contained within SEQ ID NO:103. In some embodiments of
the antibody, antigen-binding fragment, receptor, e.g., CAR,
conjugates or binding molecules provided herein, the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within SEQ ID
NO:130.
[0201] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises any of the CDR-H1,
the CDR-H2 and the CDR-H3 as described and comprises a framework
region 1 (FR1), a FR2, a FR3 and/or a FR4 having at least at or
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99%, respectively, to a FR1, a FR2, a FR3 and/or a FR4
contained within the V.sub.H region amino acid sequence set forth
in any one of SEQ ID NOs: 112, 121, 103 or 130. In some of any
embodiments, the V.sub.H region comprises a FR1, a FR2, a FR3
and/or a FR4 having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, respectively,
to a FR1, a FR2, a FR3 and/or a FR4 contained within the V.sub.H
region amino acid sequence set forth in SEQ ID NO: 112. In some of
any embodiments, the V.sub.H region comprises a FR1, a FR2, a FR3
and/or a FR4 having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, respectively,
to a FR1, a FR2, a FR3 and/or a FR4 contained within the V.sub.H
region amino acid sequence set forth in SEQ ID NO: 121. In some of
any embodiments, the V.sub.H region comprises a FR1, a FR2, a FR3
and/or a FR4 having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, respectively,
to a FR1, a FR2, a FR3 and/or a FR4 contained within the V.sub.H
region amino acid sequence set forth in SEQ ID NO: 103. In some of
any embodiments, the V.sub.H region comprises a FR1, a FR2, a FR3
and/or a FR4 having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, respectively,
to a FR1, a FR2, a FR3 and/or a FR4 contained within the V.sub.H
region amino acid sequence set forth in SEQ ID NO: 130.
[0202] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises the amino acid
sequence set forth in any one of SEQ ID NOs: 112, 121, 103 or 130.
In some embodiments of the antibody, antigen-binding fragment,
receptor, e.g., CAR, conjugates or binding molecules provided
herein, the V.sub.H region is or comprises the amino acid sequence
set forth in SEQ ID NO:112. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.H region is or
comprises the amino acid sequence set forth in SEQ ID NO:121. In
some embodiments of the antibody, antigen-binding fragment,
receptor, e.g., CAR, conjugates or binding molecules provided
herein, the V.sub.H region is or comprises the amino acid sequence
set forth in SEQ ID NO:103. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.H region is or
comprises the amino acid sequence set forth in SEQ ID NO:130.
[0203] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises the amino acid
sequence encoded by SEQ ID NO: 110, 119, 101 or 128 or a nucleic
acid sequence having at least at or about 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 110, 119, 101 or 128. In some
embodiments of the antibody, antigen-binding fragment, receptor,
e.g., CAR, conjugates or binding molecules provided herein, the
V.sub.H region is or comprises the amino acid sequence encoded by
SEQ ID NO: 111, 120, 102 or 129.
[0204] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises the amino acid
sequence encoded by SEQ ID NO: 110 or 119 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 110 or 119. In some embodiments of the
antibody, antigen-binding fragment, receptor, e.g., CAR, conjugates
or binding molecules provided herein, the V.sub.H region is or
comprises the amino acid sequence encoded by SEQ ID NO: 111 or
120.
[0205] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises the amino acid
sequence encoded by SEQ ID NO: 110 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 110. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.H region is or
comprises the amino acid sequence encoded by SEQ ID NO: 111.
[0206] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises the amino acid
sequence encoded by SEQ ID NO: 119 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 119. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.H region is or
comprises the amino acid sequence encoded by SEQ ID NO: 120.
[0207] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises the amino acid
sequence encoded by SEQ ID NO: 101 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 101. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.H region is or
comprises the amino acid sequence encoded by SEQ ID NO: 102.
[0208] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises the amino acid
sequence encoded by SEQ ID NO: 128 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 128. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.H region is or
comprises the amino acid sequence encoded by SEQ ID NO: 129. Also
provided are polynucleotides that contain any of the nucleotide
sequences described herein, e.g., encoding all of a portion of the
provided binding molecules.
[0209] In some embodiments, the antibody or antibody fragment, in
the provided CAR (e.g., an anti-ROR1 CAR), comprises a light chain
or a sufficient antigen binding portion thereof. For example, in
some embodiments, the antibody or antigen-binding fragment thereof
contains a variable light chain (V.sub.L) region, or a sufficient
antigen-binding portion of a V.sub.L region. In some embodiments,
the antibody or antigen-binding fragment thereof contains a V.sub.H
region and a variable light chain (V.sub.L) region, or a sufficient
antigen-binding portion of a V.sub.H and V.sub.L region. In any
such embodiments, a V.sub.H region sequence can be any of the
V.sub.H region sequence described herein. In any such embodiments,
a V.sub.L region sequence can be any of the V.sub.L region sequence
described herein. In any such embodiments, any of the V.sub.H
region sequence and any of the V.sub.L region sequence described
herein can be used in combination. In some of any such embodiments,
any one or more of the CDR-H1, the CDR-H2 and/or the CDR-H3
sequences described herein, and any one or more of the CDR-L1, the
CDR-L2 and/or the CDR-L3 sequences described herein can be used in
combination. In some such embodiments, the antibody is an
antigen-binding fragment, such as a Fab or an scFv. In some
embodiments, the antibody or antigen-binding fragment further
comprises at least a portion of an immunoglobulin constant region
or a variant thereof. In some such embodiments, the antibody is a
full-length antibody that also contains a constant region.
[0210] In some embodiments, a binding molecule, such as a receptor,
e.g., a CAR provided herein, contains an antibody such as an
anti-ROR1 antibody, or antigen-binding fragment thereof that
contains any of contains a V.sub.L region or a sufficient antigen
binding portion thereof. For example, in some embodiments, the CAR
contains an antibody or antigen-binding fragment thereof that
contains a V.sub.H region and a V.sub.L region, or a sufficient
antigen-binding portion of a V.sub.H and V.sub.L region. In any
such embodiments, a V.sub.H region sequence can be any of the
V.sub.H region sequence described herein. In any such embodiments,
a V.sub.L region sequence can be any of the V.sub.L region sequence
described herein. In any such embodiments, any of the V.sub.H
region sequence and any of the V.sub.L region sequence described
herein can be used in combination. In some of any such embodiments,
any one or more of the CDR-H1, the CDR-H2 and/or the CDR-H3
sequences described herein, and any one or more of the CDR-L1, the
CDR-L2 and/or the CDR-L3 sequences described herein can be used in
combination. In some such embodiments, the antibody contained in
the provided recombinant receptor is an antigen-binding fragment,
such as a Fab or an scFv. In some such embodiments, the receptor,
e.g., CAR, further contains a spacer, such as a portion of an
immunoglobulin constant region or a variant thereof, for example,
as described below in Section I.B.
[0211] In some embodiments, the V.sub.H region of an antibody or
antigen-binding fragment thereof comprises a CDR-L1, a CDR-L2
and/or a CDR-L3 according to Kabat numbering. In some embodiments,
the V.sub.H region of an antibody or antigen-binding fragment
thereof comprises a CDR-L1, a CDR-L2 and/or a CDR-L3 according to
Chothia numbering. In some embodiments, the V.sub.H region of an
antibody or antigen-binding fragment thereof comprises a CDR-L1, a
CDR-L2 and/or a CDR-L3 according to AbM numbering. In some
embodiments, the V.sub.H region of an antibody or antigen-binding
fragment thereof comprises a CDR-L1, a CDR-L2 and/or a CDR-L3
according to IMGT numbering. In some embodiments, the V.sub.H
region of an antibody or antigen-binding fragment thereof comprises
a CDR-L1, a CDR-L2 and/or a CDR-L3 according to IgBLAST numbering.
In some embodiments, the V.sub.H region of an antibody or
antigen-binding fragment thereof comprises a CDR-L1, a CDR-L2
and/or a CDR-L3 according to Kabat, Chothia, AbM, IMGT or IgBLAST
numbering, or other numbering schemes.
[0212] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, has a light chain variable (V.sub.L) region having
the amino acid sequence set forth in SEQ ID NO: 115, 124 or 106, or
an amino acid sequence that has at least at or about 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the V.sub.L region amino acid sequence set
forth in SEQ ID NO: 115, 124 or 106, or contains a light chain
complementarity determining region 1 (CDR-L1), a light chain
complementarity determining region 2 (CDR-L2) and/or a light chain
complementarity determining region 3 (CDR-L3) present in such a
V.sub.L sequence, such as one that contains a CDR-L1, a CDR-L2, and
a CDR-L3 present in such a V.sub.L sequence. In some embodiments of
the antibody, antigen-binding fragment, receptor, e.g., CAR,
conjugates or binding molecules provided herein, has a V.sub.L
region having the amino acid sequence set forth in SEQ ID NO:115 or
124, or an amino acid sequence that has at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% sequence identity to the V.sub.L region amino acid sequence set
forth in SEQ ID NO: 115 or 124, or contains a CDR-L1, a CDR-L2
and/or a CDR-L3 present in such a V.sub.L sequence, such as one
that contains a CDR-L1, a CDR-L2, and a CDR-L3 present in such a
V.sub.L sequence.
[0213] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, has a V.sub.L region having the amino acid
sequence set forth in SEQ ID NO: 115, or an amino acid sequence
that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
V.sub.L region amino acid sequence set forth in SEQ ID NO: 115, or
contains a CDR-L1, a CDR-L2 and/or a CDR-L3 present in such a
V.sub.L sequence, such as one that contains a CDR-L1, a CDR-L2, and
a CDR-L3 present in such a V.sub.L sequence. In some embodiments,
the antibody, e.g., antigen-binding fragment thereof, has a V.sub.L
region having the amino acid sequence set forth in SEQ ID NO: 124,
or an amino acid sequence that has at least at or about 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the V.sub.L region amino acid sequence set
forth in SEQ ID NO: 124, or contains a CDR-L1, a CDR-L2 and/or a
CDR-L3 present in such a V.sub.L sequence, such as one that
contains a CDR-L1, a CDR-L2, and a CDR-L3 present in such a V.sub.L
sequence. In some embodiments, the antibody, e.g., antigen-binding
fragment thereof, has a V.sub.L region having the amino acid
sequence set forth in SEQ ID NO: 106, or an amino acid sequence
that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
V.sub.L region amino acid sequence set forth in SEQ ID NO: 106, or
contains a CDR-L1, a CDR-L2 and/or a CDR-L3 present in such a
V.sub.L sequence, such as one that contains a CDR-L1, a CDR-L2, and
a CDR-L3 present in such a V.sub.L sequence.
[0214] In some of any of the provided embodiments, the V.sub.L
region comprises a CDR-L1 comprising the sequence set forth in SEQ
ID NO: 75, 90 or 60. In some of any of the provided embodiments,
the V.sub.L region comprises a CDR-L2 comprising the sequence set
forth in SEQ ID NO: 77, 92 or 62. In some of any of the provided
embodiments, the V.sub.L region comprises a CDR-L3 comprising the
sequence set forth in SEQ ID NO: 79, 94 or 64.
[0215] In some of any of the provided embodiments, the V.sub.L
region comprises a CDR-L1 comprising the sequence set forth in SEQ
ID NO: 76, 91 or 61. In some of any of the provided embodiments,
the V.sub.L region comprises a CDR-L2 comprising the sequence set
forth in SEQ ID NO: 78, 93 or 63. In some of any of the provided
embodiments, the V.sub.L region comprises a CDR-L3 comprising the
sequence set forth in SEQ ID NO: 79, 94 or 64.
[0216] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.L region comprises a CDR-L1 comprising
the sequence set forth in SEQ ID NO: 75, 90 or 60, a CDR-L2
comprising the sequence set forth in SEQ ID NO: 77, 92 or 62, and a
CDR-L3 comprising the sequence set forth in SEQ ID NO: 79, 94 or
64. In some embodiments of the antibody, antigen-binding fragment,
receptor, e.g., CAR, conjugates or binding molecules provided
herein, the V.sub.L region comprises a CDR-L1 comprising the
sequence set forth in SEQ ID NO: 76, 91 or 61, a CDR-L2 comprising
the sequence set forth in SEQ ID NO: 78, 93 or 63; and a CDR-L3
comprising the sequence set forth in SEQ ID NO: 79, 94 or 64.
[0217] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.L region comprises a CDR-L1 comprising
the sequence set forth in SEQ ID NO: 75 or 90, a CDR-L2 comprising
the sequence set forth in SEQ ID NO: 77 or 92; and a CDR-L3
comprising the sequence set forth in SEQ ID NO: 79 or 94. In some
embodiments of the antibody, antigen-binding fragment, receptor,
e.g., CAR, conjugates or binding molecules provided herein, the
V.sub.L region comprises a CDR-L1 comprising the sequence set forth
in SEQ ID NO: 76 or 91, a CDR-L2 comprising the sequence set forth
in SEQ ID NO: 78 or 93; and a CDR-L3 comprising the sequence set
forth in SEQ ID NO: 79 or 94.
[0218] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.L region comprises a CDR-L1, a CDR-L2
and a CDR-L3 comprising the sequence set forth in SEQ ID NOS:75, 77
and 79, respectively; SEQ ID NOS:90, 92 and 94; SEQ ID NOS:60, 62
and 64, respectively; or SEQ ID NOS:60, 62 and 64, respectively. In
some embodiments of the antibody, antigen-binding fragment,
receptor, e.g., CAR, conjugates or binding molecules provided
herein, the V.sub.L region comprises a CDR-L1, a CDR-L2 and a
CDR-L3 comprising the sequence set forth in SEQ ID NOS:76, 78 and
79, respectively; SEQ ID NOS:91, 93 and 94, respectively; SEQ ID
NOS:60, 63 and 64, respectively; or SEQ ID NOS:61, 63 and 64,
respectively.
[0219] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.L region comprises a CDR-L1, a CDR-L2
and a CDR-L3 comprising the sequence set forth in SEQ ID NOS:75, 77
and 79, respectively. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.L region comprises a
CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in
SEQ ID NOS:76, 78 and 79, respectively.
[0220] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.L region comprises a CDR-L1, a CDR-L2
and a CDR-L3 comprising the sequence set forth in SEQ ID NOS:90, 92
and 94, respectively. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.L region comprises a
CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in
SEQ ID NOS:91, 93 and 94, respectively.
[0221] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.L region comprises a CDR-L1, a CDR-L2
and a CDR-L3, respectively, comprising the amino acid sequence of a
CDR-L1, a CDR-L2 and a CDR-L3 contained within the V.sub.L region
amino acid sequence set forth in any one of SEQ ID NOs: 115, 124 or
106. In some embodiments of the antibody, antigen-binding fragment,
receptor, e.g., CAR, conjugates or binding molecules provided
herein, the V.sub.L region comprises a CDR-L1, a CDR-L2 and a
CDR-L3 contained within SEQ ID NO: 115 or 124.
[0222] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.L region comprises CDR-L1, a CDR-L2 and
a CDR-L3 contained within SEQ ID NO: 115. In some embodiments of
the antibody, antigen-binding fragment, receptor, e.g., CAR,
conjugates or binding molecules provided herein, the V.sub.L region
comprises CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID NO:
124. In some embodiments of the antibody, antigen-binding fragment,
receptor, e.g., CAR, conjugates or binding molecules provided
herein, the V.sub.L region comprises CDR-L1, a CDR-L2 and a CDR-L3
contained within SEQ ID NO: 106.
[0223] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.L region comprises any of the CDR-L1,
the CDR-L2 and the CDR-L3 as described and comprises a framework
region 1 (FR1), a FR2, a FR3 and/or a FR4 having at least at or
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99%, respectively, to a FR1, a FR2, a FR3 and/or a FR4
contained within the V.sub.L region amino acid sequence set forth
in any one of SEQ ID NOs: 115, 124 or 106. In some of any
embodiments, the V.sub.L region comprises a FR1, a FR2, a FR3
and/or a FR4 having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, respectively,
to a FR1, a FR2, a FR3 and/or a FR4 contained within the V.sub.L
region amino acid sequence set forth in SEQ ID NO: 115. In some of
any embodiments, the V.sub.L region comprises a FR1, a FR2, a FR3
and/or a FR4 having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, respectively,
to a FR1, a FR2, a FR3 and/or a FR4 contained within the V.sub.L
region amino acid sequence set forth in SEQ ID NO: 124. In some of
any embodiments, the V.sub.L region comprises a FR1, a FR2, a FR3
and/or a FR4 having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, respectively,
to a FR1, a FR2, a FR3 and/or a FR4 contained within the V.sub.L
region amino acid sequence set forth in SEQ ID NO: 106.
[0224] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.L region is or comprises the amino acid
sequence set forth in any one of SEQ ID NOs: 115, 124 or 106. In
some embodiments of the antibody, antigen-binding fragment,
receptor, e.g., CAR, conjugates or binding molecules provided
herein, the V.sub.L region is or comprises the amino acid sequence
set forth in SEQ ID NO: 115. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.L region is or
comprises the amino acid sequence set forth in SEQ ID NO: 124. In
some embodiments of the antibody, antigen-binding fragment,
receptor, e.g., CAR, conjugates or binding molecules provided
herein, the V.sub.L region is or comprises the amino acid sequence
set forth in SEQ ID NO: 106.
[0225] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.L region is or comprises the amino acid
sequence encoded by SEQ ID NO: 113, 122 or 104, or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 113, 122 or 104. In some embodiments of
the antibody, antigen-binding fragment, receptor, e.g., CAR,
conjugates or binding molecules provided herein, the V.sub.L region
is or comprises the amino acid sequence encoded by SEQ ID NO: 114,
123, 105 or 131.
[0226] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.L region is or comprises the amino acid
sequence encoded by SEQ ID NO: 113 or 122, or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 113 or 122. In some embodiments of the
antibody, antigen-binding fragment, receptor, e.g., CAR, conjugates
or binding molecules provided herein, the V.sub.L region is or
comprises the amino acid sequence encoded by SEQ ID NO: 114 or
123.
[0227] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.L region is or comprises the amino acid
sequence encoded by SEQ ID NO: 113, or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 113. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.L region is or
comprises the amino acid sequence encoded by SEQ ID NO: 114.
[0228] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.L region is or comprises the amino acid
sequence encoded by SEQ ID NO: 122, or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 122. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.L region is or
comprises the amino acid sequence encoded by SEQ ID NO: 123.
[0229] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.L region is or comprises the amino acid
sequence encoded by SEQ ID NO: 104, or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 104. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.L region is or
comprises the amino acid sequence encoded by SEQ ID NO: 105. In
some embodiments of the antibody, antigen-binding fragment,
receptor, e.g., CAR, conjugates or binding molecules provided
herein, the V.sub.L region is or comprises the amino acid sequence
encoded by SEQ ID NO: 131.
[0230] In some embodiments, the antibody or antigen binding
fragment is an scFv comprising a heavy chain variable (V.sub.H)
region and a light chain variable (V.sub.L) region. In some
embodiments of the antibody, antigen-binding fragment, receptor,
e.g., CAR, conjugates or binding molecules provided herein, the
V.sub.H region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 112, 121, 103 or
130, and the V.sub.L region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 115, 124
or 106. In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises an amino acid
sequence having at least at or about 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% sequence identity to SEQ ID NO:112 or 121, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% sequence identity to SEQ ID NO:115 or 124.
[0231] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:112, and the V.sub.L region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 115; the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:121, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:124; the V.sub.H
region is or comprises an amino acid sequence having at least at or
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity to SEQ ID NO:103, and the V.sub.L region
is or comprises an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:106; or the V.sub.H region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:130, and the V.sub.L region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:106.
[0232] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:112, and the V.sub.L region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 115. In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:121, and the V.sub.L region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:124. In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:103, and the V.sub.L region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:106. In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:130, and the V.sub.L region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:106.
[0233] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region of the antibody or
antigen-binding fragment thereof comprises a CDR-H1, a CDR-H2, a
CDR-H3, respectively, comprising the amino acid sequences of
CDR-H1, a CDR-H2 and a CDR-H3 contained within the V.sub.H region
amino acid sequence selected from any one of SEQ ID NOs: 112, 121,
103 or 130; and comprises a CDR-L1, a CDR-L2, a CDR-L3,
respectively, comprising the amino acid sequences of CDR-L1, a
CDR-L2 and a CDR-L3, respectively contained within the V.sub.L
region amino acid sequence selected from any one of SEQ ID NOs:
115, 124 or 106. In some of any such embodiments, the V.sub.H
region comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within
SEQ ID NO:112 or 121, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 contained within SEQ ID NO:115 or 124.
[0234] In some of any such embodiments, the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within SEQ ID
NO:112, and the V.sub.L region comprises a CDR-L1, a CDR-L2 and a
CDR-L3 contained within SEQ ID NO:115. In some of any such
embodiments, the V.sub.H region comprises a CDR-H1, a CDR-H2 and a
CDR-H3 contained within SEQ ID NO:121, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID:
NO 124. In some of any such embodiments, the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within SEQ ID
NO:103, and the V.sub.L region comprises a CDR-L1, a CDR-L2 and a
CDR-L3 contained within SEQ ID: NO 106. In some of any such
embodiments, the V.sub.H region comprises a CDR-H1, a CDR-H2 and a
CDR-H3 contained within SEQ ID NO:130, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID:
NO 106.
[0235] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a heavy chain
complementarity determining region 1 (CDR-H1) comprising the
sequence set forth in SEQ ID NO: 67, 82 or 52, a heavy chain
complementarity determining region 2 (CDR-H2) comprising the
sequence set forth in SEQ ID NO: 71, 86, 56 or 97, and a heavy
chain complementarity determining region 3 (CDR-H3) comprising the
sequence set forth in SEQ ID NO: 73, 88, 58 or 99, and the V.sub.L
region comprises a light chain complementarity determining region 1
(CDR-L1) comprising the sequence set forth in SEQ ID NO: 75, 90 or
60, a light chain complementarity determining region 2 (CDR-L2)
comprising the sequence set forth in SEQ ID NO: 77, 92 or 62; and a
light chain complementarity determining region 3 (CDR-L3)
comprising the sequence set forth in SEQ ID NO: 79, 94 or 64.
[0236] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1 comprising
the sequence set forth in SEQ ID NO: 65, 80 or 50, a CDR-H2
comprising the sequence set forth in SEQ ID NO: 69, 84, 54 or 95,
and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 73,
88, 58 or 99, and the V.sub.L region comprises a CDR-L1 comprising
the sequence set forth in SEQ ID NO: 75, 90 or 60, a CDR-L2
comprising the sequence set forth in SEQ ID NO: 77, 92 or 62; and a
CDR-L3 comprising the sequence set forth in SEQ ID NO: 79, 94 or
64; or
[0237] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1 comprising
the sequence set forth in SEQ ID NO: 66, 81 or 51, a CDR-H2
comprising the sequence set forth in SEQ ID NO: 70, 85, 55 or 96,
and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 73,
88, 58 or 99, and the V.sub.L region comprises a CDR-L1 comprising
the sequence set forth in SEQ ID NO: 75, 90 or 60, a CDR-L2
comprising the sequence set forth in SEQ ID NO: 77, 92 or 62; and a
CDR-L3 comprising the sequence set forth in SEQ ID NO: 79, 94 or
64; or
[0238] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1 comprising
the sequence set forth in SEQ ID NO: 68, 83 or 53, a CDR-H2
comprising the sequence set forth in SEQ ID NO: 72, 87, 57 or 98,
and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 74,
89, 59 or 100, and the V.sub.L region comprises a CDR-L1 comprising
the sequence set forth in SEQ ID NO: 76, 91 or 61, a CDR-L2
comprising the sequence set forth in SEQ ID NO: 78, 93 or 63; and a
CDR-L3 comprising the sequence set forth in SEQ ID NO: 79, 94 or
64.
[0239] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1 comprising
the sequence set forth in SEQ ID NO: 67 or 82, a CDR-H2 comprising
the sequence set forth in SEQ ID NO: 71 or 86, and a CDR-H3
comprising the sequence set forth in SEQ ID NO: 73 or 88, and the
V.sub.L region comprises a CDR-L1 comprising the sequence set forth
in SEQ ID NO: 75 or 90, a CDR-L2 comprising the sequence set forth
in SEQ ID NO: 77 or 92; and a CDR-L3 comprising the sequence set
forth in SEQ ID NO: 79 or 94.
[0240] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1 comprising
the sequence set forth in SEQ ID NO: 65 or 80, a CDR-H2 comprising
the sequence set forth in SEQ ID NO: 69 or 84, and a CDR-H3
comprising the sequence set forth in SEQ ID NO: 73 or 88, and the
V.sub.L region comprises a CDR-L1 comprising the sequence set forth
in SEQ ID NO: 75 or 90, a CDR-L2 comprising the sequence set forth
in SEQ ID NO: 77 or 92; and a CDR-L3 comprising the sequence set
forth in SEQ ID NO: 79 or 94.
[0241] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1 comprising
the sequence set forth in SEQ ID NO: 66 or 81, a CDR-H2 comprising
the sequence set forth in SEQ ID NO: 70 or 85, and a CDR-H3
comprising the sequence set forth in SEQ ID NO: 73 or 88, and the
V.sub.L region comprises a CDR-L1 comprising the sequence set forth
in SEQ ID NO: 75 or 90, a CDR-L2 comprising the sequence set forth
in SEQ ID NO: 77 or 92; and a CDR-L3 comprising the sequence set
forth in SEQ ID NO: 79 or 94.
[0242] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1 comprising
the sequence set forth in SEQ ID NO: 68 or 83, a CDR-H2 comprising
the sequence set forth in SEQ ID NO: 72 or 87, and a CDR-H3
comprising the sequence set forth in SEQ ID NO: 74 or 89, and the
V.sub.L region comprises a CDR-L1 comprising the sequence set forth
in SEQ ID NO: 76 or 91, a CDR-L2 comprising the sequence set forth
in SEQ ID NO: 78 or 93; and a CDR-L3 comprising the sequence set
forth in SEQ ID NO: 79 or 94.
[0243] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a heavy chain
complementarity determining region 1 (CDR-H1), a heavy chain
complementarity determining region 2 (CDR-H2) and a heavy chain
complementarity determining region 3 (CDR-H3) comprising the
sequence set forth in SEQ ID NOS:67, 71 and 73, respectively, and
the V.sub.L region comprises a light chain complementarity
determining region 1 (CDR-L1), a light chain complementarity
determining region 2 (CDR-L2) and a light chain complementarity
determining region 3 (CDR-L3) comprising the sequence set forth in
SEQ ID NOS:75, 77 and 79, respectively; the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence
set forth in SEQ ID NOS:82, 86 and 88, respectively, and the
V.sub.L region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising
the sequence set forth in SEQ ID NOS:90, 92 and 94, respectively;
the V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3
comprising the sequence set forth in SEQ ID NOS:52, 56 and 58,
respectively, and the V.sub.L region comprises a CDR-L1, a CDR-L2
and a CDR-L3 comprising the sequence set forth in SEQ ID NOS:60, 62
and 64, respectively; or the V.sub.H region comprises a CDR-H1, a
CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID
NOS:52, 97 and 99, respectively, and the V.sub.L region comprises a
CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in
SEQ ID NOS:60, 62 and 64, respectively.
[0244] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:65, 69
and 73, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:75, 77 and 79, respectively; the V.sub.H region comprises a
CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in
SEQ ID NOS:80, 84 and 88, respectively, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence
set forth in SEQ ID NOS:90, 92 and 94, respectively; the V.sub.H
region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the
sequence set forth in SEQ ID NOS:50, 54 and 58, respectively, and
the V.sub.L region comprises a CDR-L1, a CDR-L2 and a CDR-L3
comprising the sequence set forth in SEQ ID NOS:60, 62 and 64,
respectively; or the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:50, 95
and 99, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:60, 62 and 64, respectively;
[0245] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:66, 70
and 73, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:75, 77 and 79, respectively; the V.sub.H region comprises a
CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in
SEQ ID NOS:81, 85 and 88, respectively, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence
set forth in SEQ ID NOS:90, 92 and 94, respectively; the V.sub.H
region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the
sequence set forth in SEQ ID NOS:51, 55 and 58, respectively, and
the V.sub.L region comprises a CDR-L1, a CDR-L2 and a CDR-L3
comprising the sequence set forth in SEQ ID NOS:60, 62 and 64,
respectively; or the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:51, 96
and 99, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:60, 62 and 64, respectively;
[0246] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:68, 72
and 74, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:76, 78 and 79, respectively; or the V.sub.H region comprises a
CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in
SEQ ID NOS:83, 87 and 89, respectively, and the V.sub.L region
comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence
set forth in SEQ ID NOS:91, 93 and 94, respectively; the V.sub.H
region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the
sequence set forth in SEQ ID NOS:53, 57 and 59, respectively, and
the V.sub.L region comprises a CDR-L1, a CDR-L2 and a CDR-L3
comprising the sequence set forth in SEQ ID NOS:60, 63 and 64,
respectively; or the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:53, 98
and 100, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:61, 63 and 64, respectively.
[0247] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:67, 71
and 73, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:75, 77 and 79, respectively.
[0248] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:65, 69
and 73, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:75, 77 and 79, respectively.
[0249] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:66, 70
and 73, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:75, 77 and 79, respectively.
[0250] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:68, 72
and 74, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:76, 78 and 79, respectively.
[0251] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:82, 86
and 88, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:90, 92 and 94, respectively.
[0252] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:80, 84
and 88, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:90, 92 and 94, respectively.
[0253] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:81, 85
and 88, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:90, 92 and 94, respectively.
[0254] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:83, 87
and 89, respectively, and the V.sub.Lregion comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:91, 93 and 94, respectively.
[0255] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:52, 56
and 58, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:60, 62 and 64, respectively.
[0256] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:50, 54
and 58, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:60, 62 and 64, respectively.
[0257] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:51, 55
and 58, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:60, 62 and 64, respectively.
[0258] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:53, 57
and 59, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:60, 63 and 64, respectively.
[0259] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:52, 97
and 99, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:60, 62 and 64, respectively.
[0260] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:50, 95
and 99, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:60, 62 and 64, respectively.
[0261] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:51, 96
and 99, respectively, and the V.sub.Lregion comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:60, 62 and 64, respectively.
[0262] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region comprises a CDR-H1, a CDR-H2
and a CDR-H3 comprising the sequence set forth in SEQ ID NOS:53, 98
and 100, respectively, and the V.sub.L region comprises a CDR-L1, a
CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID
NOS:61, 63 and 64, respectively.
[0263] In some embodiments, the V.sub.H region of the antibody or
antigen-binding fragment thereof, receptor, e.g., CAR, conjugates
or binding molecules comprise the amino acid sequence of SEQ ID
NOs: 112, 121, 103 or 130 and the V.sub.L regions of the antibody
or antigen-binding fragment comprises the amino acid sequence 115,
124 or 106. In some embodiments, the V.sub.H and V.sub.L regions of
the antibody or antigen-binding fragment thereof, receptor, e.g.,
CAR, conjugates or binding molecules comprise the amino acid
sequences of SEQ ID NOs: 112 and 115, respectively; SEQ ID NOs: 121
and 124, respectively; SEQ ID NOs: 103 and 106, respectively; or
SEQ ID NOs: 130 and 106, respectively, or any antibody or
antigen-binding fragment thereof that has at least at or about 90%
sequence identity to any of the above V.sub.H and V.sub.L, such as
at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
or 99% sequence identity thereto. For example, the V.sub.H and
V.sub.L regions of the antibody or antigen-binding fragment thereof
provided therein comprise the amino acid sequences selected from:
SEQ ID NOs: 112 and 115; SEQ ID NOs: 121 and 124; SEQ ID NOs: 103
and 106; or SEQ ID NOs: 130 and 106, respectively.
[0264] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region and the V.sub.L region are or
comprise the sequence set forth in SEQ ID NOS: 112 and 115,
respectively. In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region and the V.sub.L region are or
comprise the sequence set forth in SEQ ID NOS: 121 and 124,
respectively. In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region and the V.sub.L region are or
comprise the sequence set forth in SEQ ID NOS: 103 and 106,
respectively. In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region and the V.sub.L region are or
comprise the sequence set forth in SEQ ID NOS: 130 and 106,
respectively.
[0265] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises the amino acid
sequence encoded by SEQ ID NO: 110, 119, 101 or 128 or a nucleic
acid sequence having at least at or about 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 110, 119, 101 or 128, and the
V.sub.L region is or comprises the amino acid sequence encoded by
SEQ ID NO: 113, 122 or 104, or a nucleic acid sequence having at
least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO: 113, 122 or 104. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the V.sub.H region is or
comprises the amino acid sequence encoded by SEQ ID NO: 111, 120,
102 or 129, and the V.sub.L region is or comprises the amino acid
sequence encoded by SEQ ID NO: 114, 123, 105 or 131.
[0266] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises the amino acid
sequence encoded by SEQ ID NO: 110 or 119 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 110 or 119, and the V.sub.L region is or
comprises the amino acid sequence encoded by SEQ ID NO: 113 or 122,
or a nucleic acid sequence having at least at or about 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 113 or 122. In
some embodiments of the antibody, antigen-binding fragment,
receptor, e.g., CAR, conjugates or binding molecules provided
herein, the V.sub.H region is or comprises the amino acid sequence
encoded by SEQ ID NO: 111 or 120, and the V.sub.L region is or
comprises the amino acid sequence encoded by SEQ ID NO: 114 or
123.
[0267] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises the amino acid
sequence encoded by SEQ ID NO: 110 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 110, and the V.sub.L region is or comprises
the amino acid sequence encoded by SEQ ID NO: 113, or a nucleic
acid sequence having at least at or about 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 113. In some embodiments of the
antibody, antigen-binding fragment, receptor, e.g., CAR, conjugates
or binding molecules provided herein, the V.sub.H region is or
comprises the amino acid sequence encoded by SEQ ID NO: 111, and
the V.sub.L region is or comprises the amino acid sequence encoded
by SEQ ID NO: 114.
[0268] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises the amino acid
sequence encoded by SEQ ID NO: 119 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 119, and the V.sub.L region is or comprises
the amino acid sequence encoded by SEQ ID NO: 122, or a nucleic
acid sequence having at least at or about 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 122. In some embodiments of the
antibody, antigen-binding fragment, receptor, e.g., CAR, conjugates
or binding molecules provided herein, the V.sub.H region is or
comprises the amino acid sequence encoded by SEQ ID NO: 120, and
the V.sub.L region is or comprises the amino acid sequence encoded
by SEQ ID NO: 123.
[0269] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises the amino acid
sequence encoded by SEQ ID NO: 101 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 101, and the V.sub.L region is or comprises
the amino acid sequence encoded by SEQ ID NO: 104, or a nucleic
acid sequence having at least at or about 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 104. In some embodiments of the
antibody, antigen-binding fragment, receptor, e.g., CAR, conjugates
or binding molecules provided herein, the V.sub.H region is or
comprises the amino acid sequence encoded by SEQ ID NO: 102 and the
V.sub.L region is or comprises the amino acid sequence encoded by
SEQ ID NO: 105.
[0270] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the V.sub.H region is or comprises the amino acid
sequence encoded by SEQ ID NO: 128 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 128 and the V.sub.L region is or comprises
the amino acid sequence encoded by SEQ ID NO: 104, or a nucleic
acid sequence having at least at or about 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 104. In some embodiments of the
antibody, antigen-binding fragment, receptor, e.g., CAR, conjugates
or binding molecules provided herein, the V.sub.H region is or
comprises the amino acid sequence encoded by SEQ ID NO: 129, and
the V.sub.L region is or comprises the amino acid sequence encoded
by SEQ ID NO: 131.
[0271] In some embodiments, the antibody or antigen-binding
fragment thereof, in the provided CAR, is a single-chain antibody
fragment, such as a single chain variable fragment (scFv) or a
diabody or a single domain antibody (sdAb). In some embodiments,
the antibody or antigen binding fragment is a multi-domain
antibody, such as an scFv comprising a heavy chain variable
(V.sub.H) region and a light chain variable (V.sub.L) region. In
some embodiments, the single-chain antibody fragment (e.g., scFv)
includes one or more linkers joining two antibody domains or
regions, such as a heavy chain variable (V.sub.H) region and a
light chain variable (V.sub.L) region. The linker typically is a
peptide linker, e.g., a flexible and/or soluble peptide linker.
Among the linkers are those rich in glycine and serine and/or in
some cases threonine. In some embodiments, the linkers further
include charged residues such as lysine and/or glutamate, which can
improve solubility. In some embodiments, the linkers further
include one or more proline.
[0272] Accordingly, the provided CARs contain anti-ROR1 antibodies
that include single-chain antibody fragments, such as scFvs and
diabodies, particularly human single-chain antibody fragments,
typically comprising linker(s) joining two antibody domains or
regions, such V.sub.H and V.sub.L regions. The linker typically is
a peptide linker, e.g., a flexible and/or soluble peptide linker,
such as one rich in glycine and serine.
[0273] In some aspects, the linkers rich in glycine and serine
(and/or threonine) include at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% such amino acid(s). In some
embodiments, they include at least at or about 50%, 55%, 60%, 70%,
or 75%, glycine, serine, and/or threonine. In some embodiments, the
linker is comprised substantially entirely of glycine, serine,
and/or threonine. The linkers generally are between about 5 and
about 50 amino acids in length, typically between at or about 10
and at or about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples
between 10 and 25 amino acids in length. Exemplary linkers include
linkers having various numbers of repeats of the sequence GGGGS
(4GS; SEQ ID NO: 39) or GGGS (3GS; SEQ ID NO: 40), such as between
2, 3, 4 and 5 repeats of such a sequence. Exemplary linkers include
those having or consisting of an sequence set forth in SEQ ID NO:
41 (GGGGSGGGGSGGGGS). Exemplary linkers further include those
having or consisting of the sequence set forth in SEQ ID NO: 24
(GSTSGSGKPGSGEGSTKG). Exemplary linkers further include those
having or consisting of the sequence set forth in SEQ ID NO: 162
(SRGGGGSGGGGSGGGGSLEMA). An exemplary linker includes those having
or consisting of the sequence set forth in SEQ ID NO; 163
(GSRGGGGSGGGGSGGGGSLEMA).
[0274] Accordingly, in some embodiments, the provided embodiments
include single-chain antibody fragments, e.g., scFvs, comprising
one or more of the aforementioned linkers, such as glycine/serine
rich linkers, including linkers having repeats of GGGS (SEQ ID NO:
40) or GGGGS (SEQ ID NO: 39), such as the linker set forth in SEQ
ID NO: 41, 162 or 163.
[0275] In some embodiments, the V.sub.H region may be amino
terminal to the V.sub.L region. In some embodiments, the V.sub.H
region may be carboxy terminal to the V.sub.L region. In particular
embodiments, the fragment, e.g., scFv, may include a V.sub.H region
or portion thereof, followed by the linker, followed by a V.sub.L
region or portion thereof. In other embodiments, the fragment,
e.g., the scFv, may include the V.sub.L region or portion thereof,
followed by the linker, followed by the V.sub.H region or portion
thereof.
[0276] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the scFv is or comprises the sequence set forth in
SEQ ID NO: 118, 127, 109 or 134, or an amino acid sequence having
at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 118,
127, 109 or 134. In some embodiments of the antibody,
antigen-binding fragment, receptor, e.g., CAR, conjugates or
binding molecules provided herein, the scFv is or comprises the
sequence set forth in SEQ ID NO: 118 or 127 or an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to
SEQ ID NO: 118 or 127. In some of any of the provided embodiments,
the scFv is or comprises the sequence set forth in SEQ ID NO: 118.
In some of any of the provided embodiments, the scFv is or
comprises the sequence set forth in SEQ ID NO: 127. In some of any
of the provided embodiments, the scFv is or comprises the sequence
set forth in SEQ ID NO: 109. In some of any of the provided
embodiments, the scFv is or comprises the sequence set forth in SEQ
ID NO: 134.
[0277] In some embodiments of the antibody, antigen-binding
fragment, receptor, e.g., CAR, conjugates or binding molecules
provided herein, the scFv is or comprises the amino acid sequence
encoded by SEQ ID NO: 116, 125, 107 or 132 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 116, 125, 107 or 132. In some of any of
the provided embodiments, the scFv is or comprises the amino acid
sequence encoded by SEQ ID NO: 117, 126, 108 or 133.
[0278] In some of any of the provided embodiments, the scFv is or
comprises the amino acid sequence encoded by SEQ ID NO: 116 or 125
or a nucleic acid sequence having at least at or about 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 116 or 125. In
some embodiments, the scFv is or comprises the amino acid sequence
encoded by SEQ ID NO: 117 or 126. In some embodiments, the scFv is
or comprises the amino acid sequence encoded by SEQ ID NO: 117. In
some embodiments, the scFv is or comprises the amino acid sequence
encoded by SEQ ID NO: 126. In some embodiments, the scFv is or
comprises the amino acid sequence encoded by SEQ ID NO: 108. In
some embodiments, the scFv is or comprises the amino acid sequence
encoded by SEQ ID NO: 133
[0279] Table 2 provides the SEQ ID NOS: of exemplary provided
antibody fragments, such as scFvs. In some aspects, the exemplary
provided antibody fragments can be comprised in the provided
ROR1-binding receptors, such as anti-ROR1 chimeric antigen
receptors (CARs). In some embodiments, the ROR1-binding antibody or
fragment thereof, such as an scFv, comprises a V.sub.H region that
comprises the CDR-H1, the CDR-H2 and the CDR-H3 sequence and a
V.sub.L region that comprises the CDR-L1, the CDR-L2 and the CDR-L3
sequence set forth in the SEQ ID NOS: listed in each row of Table 2
below (by Kabat, Chothia, AbM and IgBLAST numbering schemes). In
some embodiments, the ROR1-binding antibody or fragment thereof,
such as an scFv, comprises a V.sub.H region sequence and a V.sub.L
region sequence set forth in the SEQ ID NOS: listed in each row of
Table 2 below, or an antibody comprising a V.sub.H region and a
V.sub.L region amino acid sequence that has at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
V.sub.H region sequence and the V.sub.L region sequence set forth
in the SEQ ID NOS: listed in each row of Table 2 below. In some
embodiments, the ROR1-binding antibody or fragment thereof, such as
an scFv, comprises a V.sub.H region sequence and a V.sub.L region
sequence set forth in the SEQ ID NOS: listed in each row of Table 2
below. In some embodiments, the ROR1-binding antibody or fragment
thereof comprises an scFv sequence set forth in the SEQ ID NOS:
listed in each row of Table 2 below, or an antibody comprising an
scFv amino acid sequence that has at least at or about 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the scFv sequence set forth in the SEQ ID NOS:
listed in each row of Table 2 below. In some embodiments, the
ROR1-binding antibody or fragment thereof comprises an scFv
sequence set forth in the SEQ ID NOS: listed in each row of Table 2
below. In some embodiments, any of the antibody or antigen-binding
fragment thereof, such as scFv, listed in each row of Table 2 can
be comprised in a receptor, such as a chimeric antigen receptor
(CAR), for example, as the extracellular antigen-binding domain
[0280] In some embodiments, the provided antibody or
antigen-binding fragment thereof comprises a V.sub.H region and a
V.sub.L region, wherein the V.sub.H region of the antibody or
antigen-binding fragment thereof can contain a combination of any
of the CDR-H1, the CDR-H2 and the CDR-H3 amino acid sequences set
forth in Table 2, and the V.sub.L region of the antibody or
antigen-binding fragment thereof can contain a combination of any
of the CDR-L1, the CDR-L2 and the CDR-L3 amino acid sequences set
forth in Table 2. In some embodiments, the provided antibody or
antigen-binding fragment thereof comprises a V.sub.H region and/or
a V.sub.L region set forth in Table 2, in any combination,
orientation or containing a different linker. In some aspects, the
antibody or antigen-binding fragment thereof comprises a V.sub.H
region described in Table 2. In some aspects, the antibody or
antigen-binding fragment thereof comprises a V.sub.L region
described in Table 2. In some embodiments, the provided antibody or
antigen-binding fragment thereof is a single-domain antibody
(sdAb), comprising a V.sub.H region set forth in Table 2, or a
V.sub.L region set forth in Table 2. In some embodiments, the
provided antibody or antigen-binding fragment thereof is a
single-domain antibody (sdAb), comprising a V.sub.H region
containing a CDR-H1, a CDR-H2 and/or a CDR-H3 set forth in Table 2,
or a V.sub.L region containing a CDR-L1, a CDR-L2 and/or a CDR-L3
set forth in Table 2.
TABLE-US-00002 TABLE 2 Sequence identifier (SEQ ID NO) for
Exemplary scFvs scFv CDR-H1 CDR-H2 CDR-H3 CDR-L1 CDR-L2 CDR-L3
V.sub.H V.sub.L Linker Order scFv Kabat Kabat ROR1-1 67 71 73 75 77
79 112 115 41 V.sub.H-V.sub.L 118 ROR1-2 82 86 88 90 92 94 121 124
41 V.sub.H-V.sub.L 127 ROR1-3 52 56 58 60 62 64 103 106 41
V.sub.H-V.sub.L 109 ROR1-4 52 97 99 60 62 64 130 106 41
V.sub.H-V.sub.L 134 Chothia Chothia ROR1-1 65 69 73 75 77 79 112
115 41 V.sub.H-V.sub.L 118 ROR1-2 80 84 88 90 92 94 121 124 41
V.sub.H-V.sub.L 127 ROR1-3 50 54 58 60 62 64 103 106 41
V.sub.H-V.sub.L 109 ROR1-4 50 95 99 60 62 64 130 106 41
V.sub.H-V.sub.L 134 AbM AbM ROR1-1 66 70 73 75 77 79 112 115 41
V.sub.H-V.sub.L 118 ROR1-2 81 85 88 90 92 94 121 124 41
V.sub.H-V.sub.L 127 ROR1-3 51 55 58 60 62 64 103 106 41
V.sub.H-V.sub.L 109 ROR1-4 51 96 99 60 62 64 130 106 41
V.sub.H-V.sub.L 134 IgBLAST IgBLAST ROR1-1 68 72 74 76 78 79 112
115 41 V.sub.H-V.sub.L 118 ROR1-2 83 87 89 91 93 94 121 124 41
V.sub.H-V.sub.L 127 ROR1-3 53 57 59 61 63 64 103 106 41
V.sub.H-V.sub.L 109 ROR1-4 53 98 100 61 63 64 130 106 41
V.sub.H-V.sub.L 134
[0281] Among the provided antibodies, e.g., antigen-binding
fragments, are human antibodies. In some embodiments of a provided
human anti-ROR1 antibody, e.g., antigen-binding fragments, the
human antibody contains a V.sub.H region that comprises a portion
having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to an amino acid sequence encoded by a germline nucleotide human
heavy chain V segment, a portion having at least 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to an amino acid sequence
encoded by a germline nucleotide human heavy chain D segment,
and/or a portion having at least 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to an amino acid sequence encoded by a germline
nucleotide human heavy chain J segment; and/or contains a V.sub.L
region that comprises a portion having at least 95%, 96%, 97%, 98%,
99%, or 100% sequence identity to an amino acid sequence encoded by
a germline nucleotide human kappa or lambda chain V segment, and/or
a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to an amino acid sequence encoded by a germline nucleotide
human kappa or lambda chain J segment.
[0282] Among the provided antibodies, e.g., antigen-binding
fragments, are human antibodies. In some embodiments of a provided
human anti-ROR1 antibody, e.g., antigen-binding fragments, the
human antibody contains a V.sub.H region that comprises a portion
having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to an amino acid sequence encoded by a germline nucleotide human
heavy chain V segment, a portion having at least 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to an amino acid sequence
encoded by a germline nucleotide human heavy chain D segment,
and/or a portion having at least 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to an amino acid sequence encoded by a germline
nucleotide human heavy chain J segment; and contains a V.sub.L
region that comprises a portion having at least 95%, 96%, 97%, 98%,
99%, or 100% sequence identity to an amino acid sequence encoded by
a germline nucleotide human kappa or lambda chain V segment, and/or
a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to an amino acid sequence encoded by a germline nucleotide
human kappa or lambda chain J segment. In some embodiments, the
portion of the V.sub.H region corresponds to the CDR-H1, the CDR-H2
and/or the CDR-H3. In some embodiments, the portion of the V.sub.H
region corresponds to the CDR-H1, the CDR-H2 and the CDR-H3. In
some embodiments, the portion of the V.sub.H region corresponds to
the framework region 1 (FR1), FR2, FR2 and/or FR4. In some
embodiments, the portion of the V.sub.L region corresponds to the
CDR-L1, the CDR-L2 and/or the CDR-L3. In some embodiments, the
portion of the V.sub.L region corresponds to the CDR-L1, the CDR-L2
and the CDR-L3. In some embodiments, the portion of the V.sub.L
region corresponds to the FR1, FR2, FR2 and/or FR4.
[0283] In some embodiments, the human antibody or antigen-binding
fragment thereof, contains a CDR-H1 having at least 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to an amino acid sequence of
the corresponding CDR-H1 region within a sequence encoded by a
germline nucleotide human heavy chain V segment. For example, the
human antibody in some embodiments contains a CDR-H1 having a
sequence that is 100% identical or with no more than one, two or
three amino acid differences as compared to the corresponding
CDR-H1 region within a sequence encoded by a germline nucleotide
human heavy chain V segment.
[0284] In some embodiments, the human antibody or antigen-binding
fragment thereof, contains a CDR-H2 having at least 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to an amino acid sequence of
the corresponding CDR-H2 region within a sequence encoded by a
germline nucleotide human heavy chain V segment. For example, the
human antibody in some embodiments contains a CDR-H2 having a
sequence that is 100% identical or with no more than one, two or
three amino acid difference as compared to the corresponding CDR-H2
region within a sequence encoded by a germline nucleotide human
heavy chain V segment.
[0285] In some embodiments, the human antibody or antigen-binding
fragment thereof, contains a CDR-H3 having at least 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to an amino acid sequence of
the corresponding CDR-H3 region within a sequence encoded by a
germline nucleotide human heavy chain V segment, D segment and J
segment. For example, the human antibody in some embodiments
contains a CDR-H3 having a sequence that is 100% identical or with
no more than one, two or three amino acid differences as compared
to the corresponding CDR-H3 region within a sequence encoded by a
germline nucleotide human heavy chain V segment, D segment and J
segment.
[0286] In some embodiments, the human antibody or antigen-binding
fragment thereof, contains a CDR-L1 having at least 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to an amino acid sequence of
the corresponding CDR-L1 region within a sequence encoded by a
germline nucleotide human light chain V segment. For example, the
human antibody in some embodiments contains a CDR-L1 having a
sequence that is 100% identical or with no more than one, two or
three amino acid differences as compared to the corresponding
CDR-L1 region within a sequence encoded by a germline nucleotide
human light chain V segment.
[0287] In some embodiments, the human antibody or antigen-binding
fragment thereof, contains a CDR-L2 having at least 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to an amino acid sequence of
the corresponding CDR-L2 region within a sequence encoded by a
germline nucleotide human light chain V segment. For example, the
human antibody in some embodiments contains a CDR-L2 having a
sequence that is 100% identical or with no more than one, two or
three amino acid difference as compared to the corresponding CDR-L2
region within a sequence encoded by a germline nucleotide human
light chain V segment.
[0288] In some embodiments, the human antibody or antigen-binding
fragment thereof, contains a CDR-L3 having at least 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to an amino acid sequence of
the corresponding CDR-L3 region within a sequence encoded by a
germline nucleotide human light chain V segment and J segment. For
example, the human antibody in some embodiments contains a CDR-L3
having a sequence that is 100% identical or with no more than one,
two or three amino acid differences as compared to the
corresponding CDR-L3 region within a sequence encoded by a germline
nucleotide human light chain V segment and J segment.
[0289] In some embodiments, the human antibody or antigen-binding
fragment thereof, contains a framework region that contains human
germline gene segment sequences. For example, in some embodiments,
the human antibody contains a V.sub.H region in which the framework
region, e.g. FR1, FR2, FR3 and FR4, has at least 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to a framework region encoded
by a human germline antibody segment, such as a V segment and/or J
segment. In some embodiments, the human antibody contains a V.sub.L
region in which the framework region e.g. FR1, FR2, FR3 and FR4,
has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to
a framework region encoded by a human germline antibody segment,
such as a V segment and/or J segment. For example, in some such
embodiments, the framework region sequence contained within the
V.sub.H region and/or V.sub.L region differs by no more than 10
amino acids, such as no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino
acid, compared to the framework region sequence encoded by a human
germline antibody segment.
[0290] The antibody or antigen-binding fragment thereof, may
contain at least a portion of an immunoglobulin constant region,
such as one or more constant region domain. In some embodiments,
the constant regions include a light chain constant region and/or a
heavy chain constant region 1 (C.sub.H1). In some embodiments, the
antibody includes at least a portion of a hinge region or a variant
thereof. In some embodiments, the antibody includes a C.sub.H2
and/or C.sub.H3 domain, such as an Fc region. In some embodiments,
the Fc region is an Fc region of a human IgG, such as an IgG1 or
IgG4.
[0291] 2. Exemplary Features
[0292] In some aspects, the provided antibodies have one or more
specified functional features, such as binding properties,
including binding to particular epitopes or exhibiting lower or
reduced binding to a related but non-specific antigen. In some
aspects, the provided antibodies can bind to an epitope that is
similar to or overlaps with epitopes of other antibodies, such as
reference antibodies, and/or exhibit particular binding affinities.
In some aspects, the provided antibodies can bind to an epitope
that is different from epitopes of other antibodies, e.g., binding
a conformational epitope.
[0293] In some embodiments, the provided antibodies or
antigen-binding fragment thereof specifically bind to a receptor
tyrosine kinase-like orphan receptor 1 (ROR1) protein. In some of
any of the embodiments provided herein, ROR1 refers to human ROR1.
The observation that an antibody or other binding molecule binds to
ROR1 or specifically binds to ROR1 does not necessarily mean that
it binds to ROR1 from every species. For example, in some
embodiments, features of binding to ROR1, such as the ability to
specifically bind thereto and/or to compete for binding thereto
with a reference antibody, and/or to bind with a particular
affinity or compete to a particular degree, in some embodiments,
refers to the ability with respect to a human ROR1 protein and the
antibody may not have this feature with respect to a ROR1 of
another species such as mouse. In some embodiments, the antibody
binds to human ROR1 and binds to ROR1 of another species, such as
Rhesus macaque or cynomolgus macaque. In some embodiments, the
antibody or an antigen-binding fragment thereof binds to human ROR1
and does not bind to ROR1 of another species, such as mouse. In
some embodiments, the antibody binds to human ROR1 and binds to
ROR1 of another species, such as mouse.
[0294] In some embodiments, the antibodies, such as the anti-ROR1
antibodies, e.g., the human antibodies, specifically bind to a
particular epitope or region of ROR1, such as generally an
extracellular epitope or region. ROR1 is a type I membrane protein
that contains an extracellular region containing an immunoglobulin
(Ig) domain, a frizzled (Fz) domain and a kringle (Kr) domain
followed by a transmembrane domain. With reference to human ROR1
set forth in SEQ ID NO:144 (GenBank No. AAA60275.1; sequence
including the signal peptide set forth in SEQ ID NO:215, Uniprot
No. Q01973), the extracellular region corresponds to amino acids
1-377, amino acids 13-118 correspond to the Ig domain, amino acids
136-270 correspond to the Fz domain and amino acids 283-362
correspond to the Kr domain (corresponding to amino acids 42-147
for the Ig domain, amino acids 165-299 for the Fz domain and amino
acids 312-391 for the Kr domain with reference to amino acid
sequence set forth in SEQ ID NO:215). In some embodiments, the
antibodies, such as human antibodies, bind to an epitope comprising
residues within the Ig domain, the Fz domain and/or the Kr domain.
In some embodiments, the antibodies, such as human antibodies, bind
to an epitope comprising residues with the Ig domain and/or Fz
domain. In some embodiments, the antibodies, such as human
antibodies, bind to an epitope comprising residues within both the
Ig and Fz domains.
[0295] In some embodiments, the antibodies or antigen-binding
fragment thereof bind, such as specifically bind, to human ROR1,
such as to one or more epitopes or region of human ROR1, such as
the human ROR1 set forth in SEQ ID NO:144 (GenBank No. AAA60275.1;
sequence including the signal peptide set forth in SEQ ID NO:215,
Uniprot No. Q01973), or an allelic variant or splice variant
thereof. In some embodiments, the antibodies or antigen-binding
fragment thereof specifically binds to one or more epitopes within
a human ROR1 protein. In one embodiment, human ROR1 is a transcript
variant or isoform that has the sequence of amino acids forth in
SEQ ID NO:145 or 146. In some embodiments, human ROR1 protein
comprises an amino acid sequence set forth in SEQ ID NO: 144, 145,
146 or 215. In some embodiments, the antibodies or antigen-binding
fragment thereof bind to the extracellular region ROR1, such as to
one or more extracellular epitopes present within the extracellular
region of human ROR1, e.g., corresponding to residues 1-377 of the
human ROR1 sequence set forth in SEQ ID NO:144 (corresponding to
residues 30-406 of the human ROR1 sequence set forth in SEQ ID
NO:215 that includes the signal peptide).
[0296] In some embodiments, the antibodies or antigen-binding
fragment thereof bind one or more epitope of ROR1, such as a human
ROR1. In some embodiments, the antibodies or antigen-binding
fragment thereof bind a linear epitope of ROR1, such as a human
ROR1. In some embodiments, the antibodies or antigen-binding
fragment thereof bind one or more conformational epitopes of ROR1,
such as a human ROR1.
[0297] In some embodiments, the antibodies or antigen-binding
fragment thereof bind one or more epitopes of human ROR1, such as
one or more epitopes comprising or consisting of an amino acid
sequence selected from among any one of SEQ ID NOS: 199-214, or one
or more epitopes present within an amino acid sequence selected
from among any one of SEQ ID NOS: 199-214. In some embodiments, the
antibodies or antigen-binding fragment thereof bind one or more
epitopes of human ROR1, such as one or more epitopes that include
the sequence FRSTIYGSRLRIRNL (set forth in SEQ ID NO:199). In some
embedment's, the anti-ROR1 antibody or antigen-binding fragment
thereof specifically binds to an epitope consisting of the sequence
set forth in SEQ ID NO:199 or an epitope present within the
sequence set forth in SEQ ID NO:199. In some embodiments, the
antibodies or antigen-binding fragment thereof bind additional
epitopes, such as one or more conformational epitopes. In some
embodiments, the antibodies or antigen-binding fragment thereof
bind additional epitopes, in addition to the sequence
FRSTIYGSRLRIRNL (set forth in SEQ ID NO:199). Exemplary of one or
more additional epitopes include, but are not limited to, one or
more of the sequences set forth in SEQ ID NO:200-214 or one or more
epitopes present within an amino acid sequence selected from among
any one of SEQ ID NOS: 200-214.
[0298] In some embodiments, the antibody binds to non-human ROR1,
such as Rhesus macaques (Macaca mulatta) ROR1 (set forth in SEQ ID
NO:216, Uniprot No. F6RUP2) or cynomolgus macaques (Macaca
fasicularis) ROR1 (set forth in SEQ ID NO:217, Uniprot No.
A0A2K5WTX7; or SEQ ID NO:218, Uniprot No. A0A2K5WTX4). In some
aspects, the extracellular domain of the non-human ROR1 is at least
99% identical to the human ROR1 sequence.
[0299] In some embodiments, the antibody binds to non-human ROR1,
such as monkey, rabbit, rat, mouse, or other species of ROR1. In
some embodiments, the antibody binds to mouse (Mus musculus) ROR1,
such as to an epitope or region of mouse ROR1, such as the mouse
ROR1 set forth in SEQ ID NO: 171 (GenBank No. NP_038873; sequence
including the signal peptide set forth in SEQ ID NO:219, Uniprot
No. Q9Z139). In some embodiments, the antibody binds to human ROR1
and binds to mouse ROR1. In some embodiments, the extent of binding
of some of the provided anti-ROR1 antibodies or fragments thereof
to a non-human ROR1, such as mouse ROR1, is at least at or about
75%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150% or more of
the binding of the antibody to human ROR1.
[0300] In some of any of the provided embodiments, the antibody or
antigen-binding fragment thereof does not bind to, is not
cross-reactive to, or binds at a lower extent, level or degree or
affinity to a non-human ROR1, optionally a mouse ROR1. In some
embodiments, the extent of binding of an anti-ROR1 antibody to an
unrelated, non-ROR1 protein or to a non-human ROR1 protein, such as
a mouse ROR1 protein, or other non-ROR1 protein, is less than at or
about 50%, 40%, 30%, 20% or 10% of the binding of the antibody to
human ROR1 as measured. In some embodiments, the antibodies or
antigen-binding fragments thereof do not bind to mouse ROR1, such
as the mouse ROR1 set forth in SEQ ID NO:171 or 219. In some
embodiments, the antibodies or antigen-binding fragments do not
bind to, is not cross-reactive to, or binds at a lower level or
degree or affinity to a mouse ROR1. In some embodiments, the
extent, level or degree or affinity of binding of the provided
anti-ROR1 antibody or antigen-binding fragment thereof to a mouse
ROR1 is at least at or about 75%, 80%, 90%, 95% or 99% less than
the extent, level or degree or affinity of binding to a human
ROR1.
[0301] In some of any of the provided embodiments, the antibody or
antigen-binding fragment thereof does not bind to, is not
cross-reactive to, or binds at a lower extent, level or degree or
affinity to a receptor tyrosine kinase-like orphan receptor 2
(ROR2) protein, optionally a human ROR2 protein. In some
embodiments, the extent of binding of some of the provided
anti-ROR1 antibodies or fragments thereof to a non-ROR1 protein,
such as a ROR2 protein, is at least at or about 75%, 80%, 90%, 95%
or 99% less than the binding of the antibody to human ROR1. In some
embodiments, the provided antibody or antigen-binding fragment
thereof does not bind to, is not cross-reactive to, or binds at a
lower level or degree or affinity to a ROR2 protein, optionally a
human ROR2 protein. In some embodiments, among provided antibodies
are antibodies in which binding to mouse ROR1 is less than or at or
about 30%, 20% or 10%, such as less than at or about 10%, of the
binding of the antibody to human ROR1. In some embodiments, among
provided antibodies are antibodies in which binding to a ROR2, such
as a human ROR2, is less than or at or about 30%, 20% or 10%, such
as less than at or about 10%, of the binding of the antibody to
human ROR1.
[0302] In some embodiments, the provided antibodies are capable of
binding ROR1, such as human ROR1, with at least a certain affinity,
as measured by any of a number of known methods. In some
embodiments, the affinity is represented by an equilibrium
dissociation constant (K.sub.D). In some embodiments, the affinity
is represented by EC.sub.50.
[0303] A variety of assays are known for assessing binding
affinity, equilibrium dissociation constant (K.sub.D), equilibrium
association constant (K.sub.A), EC.sub.50, on-rate (association
rate constant; k.sub.on or k.sub.a; units of 1/Ms or
M.sup.-1s.sup.-1) and the off-rate (dissociation rate constant;
k.sub.off or k.sub.d; units of 1/s or s.sup.-1) and/or determining
whether a binding molecule (e.g., an antibody or fragment thereof)
specifically binds to a particular ligand (e.g., an antigen, such
as a ROR1 protein). One can determine the binding affinity of a
binding molecule, e.g., an antibody or an antigen-binding fragment
thereof, for an antigen, e.g., ROR1, such as human ROR1 or
cynomolgus ROR1 or mouse ROR1, such as by using any of a number of
binding assays that are well known. For example, in some
embodiments, a BIAcore.RTM. instrument can be used to determine the
binding kinetics and constants of a complex between two proteins
(e.g., an antibody or fragment thereof, and an antigen, such as a
ROR1 protein), using surface plasmon resonance (SPR) analysis (see,
e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51:660, 1949; Wilson,
Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993;
and U.S. Pat. Nos. 5,283,173, 5,468,614, or the equivalent).
[0304] SPR measures changes in the concentration of molecules at a
sensor surface as molecules bind to or dissociate from the surface.
The change in the SPR signal is directly proportional to the change
in mass concentration close to the surface, thereby allowing
measurement of binding kinetics between two molecules. The
dissociation rate constant (k.sub.off or k.sub.d), the association
rate constant (k.sub.on or k.sub.a) and/or equilibrium dissociation
constant (K.sub.D) and/or equilibrium association constant
(K.sub.A) for the complex can be determined by monitoring changes
in the refractive index with respect to time as buffer is passed
over the chip. Other suitable assays for measuring the binding of
one protein to another include, for example, immunoassays such as
enzyme linked immunosorbent assays (ELISA) and radioimmunoassays
(RIA), or determination of binding by monitoring the change in the
spectroscopic or optical properties of the proteins through
fluorescence, UV absorption, circular dichroism, or nuclear
magnetic resonance (NMR). Other exemplary assays include, but are
not limited to, Western blot, ELISA, analytical
ultracentrifugation, spectroscopy, flow cytometry, sequencing,
genetic reporter assays, flow cytometry, and other methods for
detection of expressed nucleic acids or binding of proteins.
[0305] In some embodiments, the binding molecule, e.g., antibody or
fragment thereof, binds, such as specifically binds, to an antigen,
e.g., a ROR1 protein or an epitope therein, with an affinity or
K.sub.A (i.e., an equilibrium association constant of a particular
binding interaction with units of 1/M or M.sup.-1; equal to the
ratio of the on-rate [k.sub.on or k.sub.a] to the off-rate
[k.sub.off or k.sub.d] for this association reaction, assuming
bimolecular interaction) equal to or greater than 10.sup.5
M.sup.-1. In some embodiments, the peptide binding molecule binds,
such as specifically binds, to an epitope of an antigen, e.g.,
human ROR1, with an affinity or K.sub.A (i.e., an equilibrium
association constant of a particular binding interaction with units
of 1/M or M.sup.-1) equal to or greater than 10.sup.5 M.sup.-1
(which equals the ratio of the on-rate [k.sub.on] to the off-rate
[k.sub.off] for this association reaction). In some embodiments,
the binding molecule, e.g., antibody or antigen-binding fragment
thereof, exhibits a binding affinity for a T cell epitope of the
target polypeptide with an affinity or K.sub.A ranging from at or
about 10.sup.6 M.sup.-1 to at or about 10.sup.10 M.sup.-1, such as
from at or about 10.sup.6 M.sup.-1 to at or about 10.sup.9
M.sup.-1, or from at or about 10.sup.6 M.sup.-1 to at or about
10.sup.8 M.sup.-1. In some embodiments, binding affinity may be
classified as high affinity or as low affinity. For example, in
some cases, a binding molecule, e.g., antibody or antigen-binding
fragment thereof, that exhibits high affinity binding to a
particular epitope interacts with such epitope with a K.sub.A of at
least at or about 10.sup.7 M.sup.-1, at least at or about 10.sup.8
M.sup.-1, at least at or about 10.sup.9 M.sup.-1, at least at or
about 10.sup.10 M.sup.-1, at least at or about 10.sup.-11 M.sup.-1,
at least at or about 10.sup.12 M.sup.-1, or at least at or about
10.sup.13 M.sup.-1. In some cases, a binding molecule, e.g.,
antibody or antigen-binding fragment thereof, that exhibits low
affinity binding exhibits a K.sub.A of up to 10.sup.7 M.sup.-1, up
to 10.sup.6 M.sup.-1, up to 10.sup.5 M.sup.-1.
[0306] Alternatively, affinity can be defined as an equilibrium
dissociation constant (K.sub.D) of a particular binding interaction
with units of M (e.g., 10.sup.-5 M to 10.sup.-13 M). In some
embodiments, the antibody or fragment thereof exhibits a binding
affinity for the epitope with a K.sub.D (i.e., an equilibrium
dissociation constant of a particular binding interaction with
units of M; equal to the ratio of the off-rate [k.sub.off or
k.sub.d] to the on-rate [k.sub.on or k.sub.d] for this association
reaction, assuming bimolecular interaction) of equal to or less
than 10.sup.-5 M. For example, the equilibrium dissociation
constant K.sub.D can range from 10.sup.-5 M to 10.sup.-13 M, such
as 10.sup.-7 M to 10.sup.-11 M, 10.sup.-7 M to 10.sup.-10 M,
10.sup.-7 M to 10.sup.-9 M, 10.sup.-8 M to 10.sup.-10 M, or
10.sup.-9 M to 10.sup.-10 M.
[0307] The on-rate (association rate constant; k.sub.on or k.sub.a;
units of 1/Ms or M.sup.-1 s.sup.-1) and the off-rate (dissociation
rate constant; k.sub.off or k.sub.d; units of 1/s or s.sup.-1) can
be determined using any of the known assay methods, for example,
surface plasmon resonance (SPR), or other methods described herein
for measuring the binding of one protein to another.
[0308] In some embodiments, the binding affinity (EC.sub.50) and/or
the equilibrium dissociation constant (K.sub.D) of the antibody to
ROR1, such as human ROR1, is from at or about 0.1 nM to at or about
500 nM, from at or about 0.1 nM to at or about 100 nM, from at or
about 0.1 nM to at or about 50 nM, from at or about 0.1 nM to at or
about 10 nM, from at or about 0.1 nM to at or about 1 nM, from at
or about 1 nM to at or about 500 nM, from at or about 1 nM to at or
about 100 nM, from at or about 1 nM to at or about 50 nM, from at
or about 1 nM to at or about 10 nM, from at or about 10 nM to at or
about 500 nM, from at or about 10 nM to at or about 100 nM, from at
or about 10 nM to at or about 50 nM, from at or about 50 nM to at
or about 500 nM, from at or about 50 nM to at or about 100 nM or
from at or about 100 nM to at or about 500 nM. In certain
embodiments, the binding affinity (EC.sub.50) and/or the
equilibrium dissociation constant (K.sub.D) of the antibody to
ROR1, such as human ROR1, is at or about or less than at or about
100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16
nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6
nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM, or a range defined by any of
the foregoing. In some embodiments, the antibodies bind to ROR1,
such as human ROR1, with a sub-nanomolar binding affinity, for
example, with a binding affinity less than at or about 1 nM, such
as less than at or about 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM,
0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM. In some embodiments, the binding
affinity (EC.sub.50) and/or the equilibrium dissociation constant,
K.sub.D, of the binding molecule, e.g., anti-ROR1 antibody or
fragment thereof, to a ROR1 protein, such as a human ROR1 protein,
is from at or about 0.01 nM to about 1 .mu.M, 0.1 nM to 1 .mu.M, 1
nM to 1 .mu.M, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM
to 10 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 50 nM
to 500 nM, 50 nM to 100 nM or 100 nM to 500 nM. In certain
embodiments, the binding affinity (EC.sub.50) and/or the
equilibrium dissociation constant, K.sub.D, of the binding
molecule, e.g., anti-ROR1 antibody or fragment thereof, to a ROR1
protein, such as a human ROR1 protein, is at or about or less than
at or about 1 .mu.M, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20
nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM,
10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or
less, or a range defined by any of the foregoing.
[0309] In some embodiments, the EC.sub.50 and/or the K.sub.D of the
binding molecule, e.g., anti-ROR1 antibody or fragment thereof, to
a ROR1 protein, is between at or about 10 nM and at or about 90 nM,
between at or about 20 nM and at or about 80 nM, between at or
about 30 nM and at or about 70 nM, between at or about 40 nM and at
or about 60 nM, or between at or about 40 nM and at or about 50 nM.
In certain embodiments, the EC.sub.50 and/or the K.sub.D of the
binding molecule, e.g., anti-ROR1 antibody or fragment thereof, to
a ROR1 protein, such as a human ROR1 protein, is at or about 10 nM,
20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM or 100 nM,
or a range defined by any of the foregoing. In certain embodiments,
the EC.sub.50 and/or the K.sub.D of the binding molecule, e.g.,
anti-ROR1 antibody or fragment thereof, to a ROR1 protein, such as
a human ROR1 protein, is at or about 10 nM, 20 nM, 30 nM, 40 nM, 50
nM, 60 nM, 70 nM, 80 nM, 90 nM or 100 nM, or a range defined by any
of the foregoing. In certain embodiments, the EC.sub.50 and/or the
K.sub.D of the binding molecule, e.g., anti-ROR1 antibody or
fragment thereof, to a ROR1 protein, such as a human ROR1 protein,
is at or about 40 nM, 41 nM, 42 nM, 43 nM, 44 nM, or 45 nM, or a
range defined by any of the foregoing.
[0310] In some embodiments, the V.sub.H region of the provided
binding molecules comprises a CDR-H1, a CDR-H2 and a CDR-H3
contained within SEQ ID NO:112, and the V.sub.L region of the
provided binding molecules comprises a CDR-L1, a CDR-L2 and a
CDR-L3 contained within SEQ ID: NO 115 and the equilibrium
dissociation constant (K.sub.D) for human ROR1 is between at or
about 30 nM and at or about 50 nM. In some embodiments, the V.sub.H
region and the V.sub.L region of the provided binding molecules are
or comprise the sequence set forth in SEQ ID NOS: 112 and 115,
respectively, and the off-rate (k.sub.off or k.sub.d) for human
ROR1 is at or about 40 nM.
[0311] In some embodiments, the provided binding molecule, e.g.,
anti-ROR1 antibody or antigen-binding fragment thereof or receptors
containing such antibody or antigen-binding fragments, has a fast
off-rate (dissociation rate constant; k.sub.off or Ica; units of
1/s or s.sup.-1). In some embodiments, the off-rate (k.sub.off or
k.sub.d) of the provided binding molecules is between at or about
1.times.10.sup.-5 s.sup.-1 and at or about 1.times.10.sup.-2
s.sup.-1, such as at or about 5.times.10.sup.-5 s.sup.-1 and at or
about 9.times.10.sup.-3 at or about 1.times.10 s.sup.-1 and at or
about 8.times.10.sup.-3 s.sup.-1, at or about 5.times.10.sup.-4
s.sup.-1 and at or about 7.times.10.sup.-3 s.sup.-1, at or about
1.times.10.sup.-3 s.sup.-1 and at or about 6.times.10.sup.-3
s.sup.-1, and at or about 4.times.10.sup.-3 s.sup.-1 and at or
about 6.times.10.sup.-3 s.sup.-1. In some embodiments, the off-rate
(k.sub.off or k.sub.d) of the provided binding molecules is at
least at or about 1.times.10.sup.-5 s.sup.-1, 5.times.10.sup.-5
s.sup.-1, 1.times.10.sup.-4 s.sup.-1, 5.times.10.sup.-4 s.sup.-1,
1.times.10.sup.-3 s.sup.-1, 5.times.10.sup.-3 s.sup.-1, or
1.times.10.sup.-2 s.sup.-1. In some embodiments, the off-rate
(k.sub.off or k.sub.d) of the provided binding molecules is at
least at or about 6.times.10.sup.-4 s.sup.-1, 7.times.10.sup.-4
s.sup.-1, 8.times.10.sup.-4 s.sup.-1, 9.times.10.sup.-4 s.sup.-1,
1.times.10.sup.-3 s.sup.-1, 2.times.10.sup.-3 s.sup.-1,
3.times.10.sup.-3 s.sup.-1, 4.times.10.sup.-3 s.sup.-1,
5.times.10.sup.-3 s.sup.-1, 6.times.10.sup.-3 s.sup.-1,
7.times.10.sup.-3 s.sup.-1, 8.times.10.sup.-3 s.sup.-1,
9.times.10.sup.-3 s.sup.-1 or 1.times.10.sup.-2 s.sup.-1. In some
embodiments, the off-rate (k.sub.off or k.sub.d) of the provided
binding molecules is at least at or about 4.times.10.sup.-3
s.sup.-1, 5.times.10.sup.-3 s.sup.-1 or 6.times.10.sup.-3 s.sup.-1,
or a range defined by any of the foregoing. In some embodiments,
the provided binding molecule, e.g., anti-ROR1 antibody or
antigen-binding fragment thereof or receptors containing such
antibody or antigen-binding fragments, has an off-rate that is at
least at or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,
8-fold, 9-fold or 10-fold faster than the off-rate of a reference
anti-ROR1 antibody or an antigen-binding fragment thereof, or
receptors containing such antibody or antigen-binding fragments,
for example, anti-ROR1 antibody R12.
[0312] In some embodiments, the V.sub.H region of the provided
binding molecules comprises a CDR-H1, a CDR-H2 and a CDR-H3
contained within SEQ ID NO:112, and the V.sub.L region of the
provided binding molecules comprises a CDR-L1, a CDR-L2 and a
CDR-L3 contained within SEQ ID: NO 115 and the off-rate (k.sub.off
or k.sub.d) for human ROR1 is between at or about 4.times.10.sup.-3
s.sup.-1 and at or about 6.times.10.sup.-3 s.sup.-1. In some
embodiments, the V.sub.H region and the V.sub.L region of the
provided binding molecules are or comprise the sequence set forth
in SEQ ID NOS: 112 and 115, respectively, and the off-rate
(k.sub.off or k.sub.d) for human ROR1 is at or about
5.times.10.sup.-3 s.sup.-1.
[0313] In some embodiments, the binding affinity of a binding
molecule, such as an anti-ROR1 antibody, for different antigens,
e.g., ROR1 proteins from different species can be compared to
determine the species cross-reactivity. For example, species
cross-reactivity can be classified as high cross reactivity or low
cross reactivity. In some embodiments, the equilibrium dissociation
constant, K.sub.D, for different antigens, e.g., ROR1 proteins from
different species such as human, cynomolgus monkey or mouse, can be
compared to determine species cross-reactivity. In some
embodiments, the species cross-reactivity of an anti-ROR1 antibody
can be high, e.g., the anti-ROR1 antibody binds to human ROR1 and a
species variant ROR1 to a similar degree, e.g., the ratio of
K.sub.D for human ROR1 and K.sub.D for the species variant ROR1 is
or is about 1. In some embodiments, the species cross-reactivity of
an anti-ROR1 antibody can be low, e.g., the anti-ROR1 antibody has
a high affinity for human ROR1 but a low affinity for a species
variant ROR1, or vice versa. For example, the ratio of K.sub.D for
the species variant ROR1 and K.sub.D for the human ROR1 is more
than 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500,
1000, 2000 or more, and the anti-ROR1 antibody has low species
cross-reactivity. The degree of species cross-reactivity can be
compared with the species cross-reactivity of a known antibody,
such as a reference antibody.
[0314] In some embodiments, the provided antibodies or antigen
binding fragments thereof bind to a similar degree to a human ROR1
protein and a non-human ROR1 protein. For example, in some
embodiments, the provided antibodies or antigen binding fragments
thereof bind to a human ROR1 protein, or an allelic variant or
splice variant thereof, with a specific an equilibrium dissociation
constant (K.sub.D), and to a non-human ROR1, such as a cynomolgus
monkey ROR1, with a Kr) that is similar, or about the same, or less
than 2-fold different, or less than 5-fold different.
[0315] For example, in some embodiments, the provided antibodies or
antigen binding fragments thereof bind to a human ROR1 with a
K.sub.D of about or less than at or about 1 .mu.M, 500 nM, 100 nM,
50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15
nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5
nM, 4 nM, 3 nM, 2 nM, or 1 nM or less, and binds to a cynomolgus
monkey ROR1 with a K.sub.D of about or less than at or about 1
.mu.M, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18
nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8
nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less. In some
embodiments, the provided antibodies or antigen binding fragments
thereof bind to a mouse ROR1 protein with a K.sub.D of about or
less than at or about 1 .mu.M, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM,
25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12
nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM,
or 1 nM or less. In some embodiments, the provided antibodies or
antigen binding fragments thereof bind to a human ROR1, a
cynomolgus monkey ROR1 and a mouse ROR1 with high affinity. In some
embodiments, the provided antibodies or antigen binding fragments
thereof bind to a human ROR1 and cynomolgus monkey ROR1 with a high
affinity, and to a mouse ROR1 with low affinity. In some
embodiments, the provided antibodies or antigen binding fragments
thereof bind to a human ROR1 and ROR1 from other species, or other
variants of the ROR1 protein, with high affinity.
[0316] In some embodiments, the total binding capacity (R.sub.max),
as measured using particular surface plasmon resonance (SPR)
conditions, is used to determine the ability or capacity of binding
of the provided antibody or antigen binding fragment thereof, to
the antigen, e.g., a ROR1 protein, such as a human ROR1 protein.
For SPR analysis, the "ligand" is the immobilized target molecule
on the surface of the sensor, for example, a ROR1 protein, and the
"analyte" is the tested molecule, e.g., antibody, for binding to
the "ligand". For example, the "analyte" can be any of the provided
antibodies or antigen binding fragments thereof, that binds to a
ROR1 protein. For a particular ligand and analyte pair in SPR, the
R.sub.max can be determined assuming a 1:1 binding stoichiometry
model, for a particular condition. In some embodiments, binding
capacity (R.sub.max) can be determined using the following formula:
R.sub.max (RU)=(analyte molecular weight)/(ligand molecular
weight).times.immobilized ligand level (RU). In particular aspects
of SPR conditions, the R.sub.max of binding between any of the
provided antibody or antigen binding fragment thereof and a ROR1
protein, such as a human ROR1 or a cynomolgus ROR1, is at least or
at least about 50 resonance units (RU), such as about 25 RU, 20 RU,
15 RU, 10 RU, 5 RU or 1 RU.
[0317] In some embodiments, properties or features of the provided
antibodies are described in relation to properties observed for
another antibody, e.g., a reference antibody. In some embodiments,
the reference antibody is a non-human anti-ROR1 antibody, such as a
rabbit or chimeric or humanized anti-ROR1 antibody. In some
aspects, the reference antibody is the chimeric rabbit/human IgG1
antibody designated R12 (see, e.g., Yang et al. (2011) PloS ONE,
6:e21018; U.S. Patent Application No. US 2013/0251642), and/or a
fragment derived therefrom such as an scFv fragment thereof, and/or
an antibody containing the V.sub.H and V.sub.L sequences of such an
antibody and/or the heavy and light chain CDRs of such an antibody.
A chimeric antigen receptor (CAR) containing an antigen-binding
scFv fragment of R12 has been demonstrated to effectively promote
antitumor reactivity in a CAR therapy (Hudecek et al. (2013) Clin.
Cancer Res., 19:3153; International published PCT Appl. No.
WO2014031687). In some embodiments, the reference antibody is an
scFv that comprises the sequence of amino acids set forth in SEQ ID
NO:142.
[0318] In some embodiments, the reference antibody is the mouse
anti-human ROR1 antibody designated 2A2, and/or a fragment derived
therefrom such as an scFv fragment thereof, and/or an antibody
containing the V.sub.H and V.sub.L sequences of such an antibody
and/or the heavy and light chain CDRs of such an antibody (see,
e.g., Baskar et al. (2012) MAbs, 4:349-361; published U.S. Patent
Appl. No. US2012/20058051). For example, in some embodiments, the
reference antibody has a V.sub.H region containing the sequence set
forth in SEQ ID NO: 172 and a V.sub.L containing the sequence set
forth in SEQ ID NO: 173. In some embodiments, the reference
antibody is an scFv form of antibody 2A2.
[0319] In some embodiments, the reference antibody is a human or
humanized anti-ROR1 antibody. Exemplary humanized anti-ROR1
antibodies are described in International PCT Appl. No.
WO2014/031174. In some embodiments, the reference antibody is a
humanized variant of an antibody designated 99961. In some
embodiments, the reference antibody has a V.sub.H region containing
the sequence set forth in SEQ ID NO: 174, 175, 176 or 177 and a
V.sub.L containing the sequence set forth in SEQ ID NO: 178, 179,
180 or 181.
[0320] In some embodiments, the provided antibody contains heavy
and light chain CDRs that are distinct from the CDRs present in the
reference antibody or antibodies. Among the provided antibodies are
those that compete for binding with and/or bind to the same or
overlapping epitopes of ROR1 as those bound by a reference antibody
or antibody, such as R12, but nonetheless contain distinct CDRs,
e.g., distinct heavy and/or light chain CDR1, CDR2, and CDR3.
[0321] In some embodiments, the antibody has an affinity that is
about the same as or lower than that of the corresponding form of
the reference antibody, e.g., EC.sub.50 or K.sub.D that is no more
than at or about 1.5-fold or no more than at or about 2-fold
greater, no more than at or about 3-fold greater, and/or no more
than at or about 10-fold greater, than the EC.sub.50 or K.sub.D of
the corresponding form of the reference antibody. In some
embodiments, the antibody has an affinity that is about the same as
or lower than that of the corresponding form of the reference
antibody, e.g., EC.sub.50 or K.sub.D that is at least at or about
1.5-fold greater, at least at or about 2-fold greater, at least at
or about 3-fold greater, at least at or about 5-fold greater, at
least at or about 10-fold greater, at least at or about 20-fold
greater, at least at or about 25-fold greater, at least at or about
30-fold greater, at least at or about 40-fold greater, at least at
or about 50-fold greater, or at least at or about 100-fold greater,
than the EC.sub.50 or K.sub.D of the corresponding form of the
reference antibody. In some embodiments, the antibody has an
affinity that is about the same as or lower than that of the
corresponding form of the reference antibody, an affinity that is
at or about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,
7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 30-fold,
40-fold, 50-fold or 100-fold lower than the affinity of the
reference antibody.
[0322] In some embodiments, the antibody has an affinity that is
greater than that of the corresponding form of the reference
antibody, e.g., EC.sub.50 or K.sub.D that is lower than or lower
than at or about 5-fold, 10-fold, 20-fold, 30-fold, 40-fold,
50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold,
200-fold, 250-fold or lower than the EC.sub.50 or K.sub.D of the
corresponding form of the reference antibody.
[0323] In some embodiments, the antibodies display a binding
preference for ROR1-expressing cells as compared to ROR1-negative
cells, such as particular cells known and/or described herein to
express ROR1 and known not to express ROR1, or expressing a related
but different antigen, e.g., ROR2. In some embodiments, the binding
preference is observed where a significantly greater degree of
binding is measured to the ROR1-expressing, as compared to the
non-expressing, cells or cells expressing a related but different
antigen. In some embodiments, the fold change in degree of binding
detected, for example, as measured by mean fluorescence intensity
in a flow cytometry-based assay and/or dissociation constant or
EC.sub.50, to the ROR1-expressing cells as compared to the
non-ROR1-expressing cells or cells expressing a related but
different antigen, is at least at or about 1.5, 2, 3, 4, 5, 6, or
more, and/or is about as great, about the same, at least as great
or at least about as great, or greater, than the fold change
observed for the corresponding form of the reference antibody. In
some cases, the total degree of observed binding to ROR1 or to the
ROR1-expressing cells is approximately the same, at least as great,
or greater than that observed for the corresponding form of the
reference antibody.
[0324] In some aspects, the affinity is at or about the same degree
or substantially the same degree of affinity compared to the
corresponding form of the reference antibody, such as rabbit ROR1
antibody. In some aspects, the affinity is at least at or about 80,
85, 90, 95, or 99% of or the same as that of the corresponding form
of the reference antibody.
[0325] In some embodiments, the antibody specifically binds to an
epitope that overlaps with the epitope of ROR1 bound by a reference
antibody. In some aspects, among such antibodies are antibodies
that bind to the same or a similar epitope as the reference
antibody. In some embodiments, the antibodies bind to the same or a
similar epitope or an epitope within the same region or containing
residues within the same region of ROR1 as a reference antibody,
such as anti-ROR1 antibody R12 or scFv fragment thereof (set forth
in SEQ ID NO: 142; see e.g. Yang et al. (2011) PloS ONE, 6:e21018).
In some embodiments, the antibody inhibits binding to and/or
competes for binding to ROR1, such as human ROR1, with the
reference antibody.
[0326] Competitive inhibition assays are known and include
ELISA-based, flow cytometry-based assays, and RIA-based assays. In
some aspects, competitive inhibition assays are carried out by
incorporating an excess of an unlabeled form of one of the
antibodies and assessing its ability to block binding of the other
antibody, which is labeled with a detectable marker, such that
degree of binding and reduction thereof can be assessed by
detection of the label or marker. In some embodiments, addition of
the provided antibody in excess, e.g., 1-, 2-, 5-, 10-, 50- or
100-fold excess, as compared to the amount or concentration of the
reference antibody, inhibits binding to the antigen by the
reference antibody (or vice versa). In some embodiments, the
inhibition of binding is by at least 50%, and in some embodiments
by at least 75%, 90% or 99%. In some aspects, the competitive
inhibition is as measured in a competitive binding assay (see,
e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Competition
assays may be used to identify an antibody that competes with any
of the antibodies described herein. Assays for mapping epitopes
bound by the antibodies and reference antibodies also may be used
and are known.
[0327] In some embodiments, where the reference antibody is present
at a concentration of or of about 2 nM, the provided antibody
inhibits binding of the reference antibody with an IC.sub.50 of
less than at or about 200 nM, 150 nM, 100 nM, 50 nM, 40 nM, 30 nM,
25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12
nM, 11 nM, or 10 nM, or less than at or about 9 nM, 8 nM, 7 nM, 6
nM, or 5 nM. In some embodiments, where the provided antibody is
present at a concentration of or about 2 nM, the reference antibody
inhibits binding of the provided antibody with an IC.sub.50 of less
than at or about 200 nM, 150 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25
nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM,
11 nM, or 10 nM, or less than at or about 9 nM, 8 nM, 7 nM, 6 nM,
or 5 nM. In some embodiments, competitive inhibition of the
reference antibody's binding by the provided antibody (or vice
versa) is at or about or least at or about the same degree as the
degree of competitive inhibition of the reference antibody's
binding by the reference antibody itself, e.g., unlabeled reference
antibody. In some embodiments, the provided antibody inhibits
binding of the reference antibody, such as binding of R12 scFv, to
human ROR1 by at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99%.
[0328] Anti-ROR1 antibodies provided herein may be identified,
screened for, or characterized for their physical/chemical
properties and/or biological activities by various known assays. In
one aspect, the antibody is tested for its antigen binding
activity, e.g., by known methods such as ELISA, Western blotting,
and/or flow cytometric assays, including cell-based binding assays,
for example, assessing binding of the antibody (e.g., conjugated to
a fluorescent marker or tagged) to a cell expressing the target
antigen, e.g., ROR1, in some cases compared to results using cells
that do not express the target antigen, e.g., ROR1, or cells that
express a different antigen, e.g., ROR2. Binding affinity may be
measured as K.sub.D, K.sub.A or EC.sub.50.
[0329] 3. Variants
[0330] In certain embodiments, the antibodies include one or more
amino acid variations, e.g., substitutions, deletions, insertions,
and/or mutations, compared to the sequence of an antibody described
herein. Exemplary variants include those designed to improve the
binding affinity and/or other biological properties of the antibody
Amino acid sequence variants of an antibody may be prepared by
introducing appropriate modifications into the nucleotide sequence
encoding the antibody, or by peptide synthesis. Such modifications
include, for example, deletions from, and/or insertions into and/or
substitutions of residues within the amino acid sequences of the
antibody. Any combination of deletion, insertion, and substitution
can be made to arrive at the final construct, provided that the
final construct possesses the desired characteristics, e.g.,
antigen-binding.
[0331] In certain embodiments, the antibodies include one or more
amino acid substitutions, e.g., as compared to an antibody sequence
described herein and/or compared to a sequence of a natural
repertoire, e.g., human repertoire. Sites of interest for
substitutional mutagenesis include the CDRs and FRs Amino acid
substitutions may be introduced into an antibody of interest and
the products screened for a desired activity, e.g.,
retained/improved antigen binding, decreased immunogenicity,
improved half-life, and/or improved effector function, such as the
ability to promote antibody-dependent cellular cytotoxicity (ADCC)
or complement-dependent cytotoxicity (CDC).
[0332] In some embodiments, one or more residues within a CDR of a
parent antibody (e.g. a humanized or human antibody) is/are
substituted. In some embodiments, the substitution is made to
revert a sequence or position in the sequence to a germline
sequence, such as an antibody sequence found in the germline (e.g.,
human germline), for example, to reduce the likelihood of
immunogenicity, e.g., upon administration to a human subject.
[0333] In some embodiments, alterations are made in CDR "hotspots,"
residues encoded by codons that undergo mutation at high frequency
during the somatic maturation process (see, e.g., Chowdhury,
Methods Mol. Biol. 207:179-196 (2008)), and/or residues that
contact antigen, with the resulting variant V.sub.H or V.sub.L
being tested for binding affinity. Affinity maturation by
constructing and reselecting from secondary libraries has been
described, e.g., in Hoogenboom et al. in Methods in Molecular
Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J.,
(2001)). In some embodiments of affinity maturation, diversity is
introduced into the variable genes chosen for maturation by any of
a variety of methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves CDR-directed approaches, in which several CDR
residues (e.g., 4-6 residues at a time) are randomized. CDR
residues involved in antigen binding may be specifically
identified, e.g., using alanine scanning mutagenesis or modeling.
CDR-H3 and CDR-L3 in particular are often targeted.
[0334] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more CDRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in CDRs. Such
alterations may, for example, be outside of antigen contacting
residues in the CDRs. In certain embodiments of the variant V.sub.H
and V.sub.L sequences provided above, each CDR either is unaltered,
or contains no more than one, two or three amino acid
substitutions.
[0335] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme or a polypeptide which increases the serum
half-life of the antibody.
[0336] 4. Modifications
[0337] In certain embodiments, the antibody is altered to increase
or decrease the extent to which the antibody is glycosylated, for
example, by removing or inserting one or more glycosylation sites
by altering the amino acid sequence and/or by modifying the
oligosaccharide(s) attached to the glycosylation sites, e.g., using
certain cell lines.
[0338] In some embodiments, an N-linked glycosylation, which is a
glycosylation site that occurs at asparagines in the consensus
sequence -Asn-Xaa-Ser/Thr is removed or inserted. In some
embodiments, one or more re replaced with another amino acid to
remove the glycosylation site.
[0339] Exemplary modifications, variants, and cell lines are
described, e.g., in Patent Publication Nos. US 2003/0157108, US
2004/0093621, US 2003/0157108; WO 2000/61739; WO 2001/29246; US
2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US
2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO
2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742;
WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004);
Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Ripka et al.
Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US
2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Yamane-Ohnuki et
al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol.
Bioeng., 94(4):680-688 (2006); and WO2003/085107); WO 2003/011878
(Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and
US 2005/0123546 (Umana et al.); WO 1997/30087 (Patel et al.); WO
1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
[0340] Among the modified antibodies are those having one or more
amino acid modifications in the Fc region, such as those having a
human Fc region sequence or other portion of a constant region
(e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an
amino acid modification (e.g. a substitution) at one or more amino
acid positions.
[0341] Such modifications can be made, e.g., to improve half-life,
alter binding to one or more types of Fc receptors, and/or alter
effector functions.
[0342] Also among the variants are cysteine engineered antibodies
such as "thioMAbs" and other cysteine engineered variants, in which
one or more residues of an antibody are substituted with cysteine
residues, in order to generate reactive thiol groups at accessible
sites, e.g., for use in conjugation of agents and linker-agents, to
produce immunoconjugates. Cysteine engineered antibodies are
described, e.g., in U.S. Pat. Nos. 7,855,275 and 7,521,541.
[0343] In some embodiments, the antibodies are modified to contain
additional nonproteinaceous moieties, including water soluble
polymers. Exemplary polymers include, but are not limited to,
polyethylene glycol (PEG), copolymers of ethylene glycol/propylene
glycol, carboxymethylcellulose, dextran, polyvinyl alcohol,
polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer is attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0344] B. Immunoconjugates
[0345] In some embodiments, the antibody is or is part of an
immunoconjugate, in which the antibody is conjugated to one or more
heterologous molecule(s), such as, but not limited to, a cytotoxic
or an imaging agent. Cytotoxic agents include, but are not limited
to, radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,
Pb.sup.212 and radioactive isotopes of Lu); chemotherapeutic agents
(e.g., methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents); growth
inhibitory agents; enzymes and fragments thereof such as
nucleolytic enzymes; antibiotics; toxins such as small molecule
toxins or enzymatically active toxins. In some embodiments, the
antibody is conjugated to one or more cytotoxic agents, such as
chemotherapeutic agents or drugs, growth inhibitory agents, toxins
(e.g., protein toxins, enzymatically active toxins of bacterial,
fungal, plant, or animal origin, or fragments thereof), or
radioactive isotopes.
[0346] Among the immunoconjugates are antibody-drug conjugates
(ADCs), in which an antibody is conjugated to one or more drugs,
including but not limited to a maytansinoid (see U.S. Pat. Nos.
5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an
auristatin such as monomethylauristatin drug moieties DE and DF
(MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and
7,498,298); a dolastatin; a calicheamicin or derivative thereof
(see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,
5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al.,
Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res.
58:2925-2928 (1998)); an anthracycline such as daunomycin or
doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523
(2006); Jeffrey et al., Bioorganic & Med. Chem. Letters
16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005);
Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000);
Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532
(2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S.
Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as
docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a
trichothecene; and CC1065.
[0347] Also among the immunoconjugates are those in which the
antibody is conjugated to an enzymatically active toxin or fragment
thereof, including but not limited to diphtheria A chain,
nonbinding active fragments of diphtheria toxin, exotoxin A chain
(from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
Momordica charantia inhibitor, curcin, crotin, Sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes.
[0348] Also among the immunoconjugates are those in which the
antibody is conjugated to a radioactive atom to form a
radioconjugate. Exemplary radioactive isotopes include At.sup.211,
I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of
Lu.
[0349] Conjugates of an antibody and cytotoxic agent may be made
using any of a number of known protein coupling agents, e.g.,
linkers, (see Vitetta et al., Science 238:1098 (1987)), WO94/11026.
The linker may be a "cleavable linker" facilitating release of a
cytotoxic drug in the cell, such as acid-labile linkers,
peptidase-sensitive linkers, photolabile linkers, dimethyl linkers,
and disulfide-containing linkers (Chari et al., Cancer Res.
52:127-131 (1992); U.S. Pat. No. 5,208,020).
[0350] C. Multispecific Antibodies
[0351] In certain embodiments, the ROR1-binding molecules, e.g.,
antibodies or polypeptides such as chimeric receptors containing
the same, are multispecific. Among the multispecific binding
molecules are multispecific antibodies, including, e.g. bispecific.
Multispecific binding partners, e.g., antibodies, have binding
specificities for at least two different sites, which may be in the
same or different antigens. In certain embodiments, one of the
binding specificities is for ROR1 and the other is for another
antigen. In certain embodiments, bispecific antibodies may bind to
two different epitopes of ROR1. Bispecific antibodies may also be
used to localize cytotoxic agents to cells which express ROR1.
Bispecific antibodies can be prepared as full length antibodies or
antibody fragments. Among the multispecific antibodies are
multispecific single-chain antibodies, e.g., diabodies, triabodies,
and tetrabodies, tandem di-scFvs, and tandem tri-scFvs. Also
provided are multispecific chimeric receptors, such as
multispecific CARs, containing the antibodies. Also provided are
multispecific cells containing the antibodies or polypeptides
including the same, such as cells containing a cell surface protein
including the anti-ROR1 antibody and an additional cell surface
protein, such as an additional chimeric receptor, which binds to a
different antigen or a different epitope on ROR1.
[0352] Exemplary additional antigens include B cell specific
antigens, other tumor-specific antigens, such as antigens expressed
specifically on or associated with B cell leukemia, lymphoma, B
cell chronic lymphocytic leukemia (CLL), acute myeloid leukemia
(AML), acute lymphocytic leukemia (ALL), Burkitt's Lymphoma, mantle
cell lymphoma (MCL), non-small cell lung cancer (NSCLC),
neuroblastoma, renal cell carcinoma, colon cancer, colorectal
cancer, breast cancer, epithelial squamous cell cancer, melanoma,
myeloma, stomach cancer, brain cancer, lung cancer, pancreatic
cancer, cervical cancer, ovarian cancer, liver cancer, bladder
cancer, prostate cancer, testicular cancer, thyroid cancer, uterine
cancer, adrenal cancer and/or head and neck cancer, and antigens
expressed on T cells. Exemplary antigens include CD4, CD5, CD8,
CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38,
CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC-1,
Ia, HM1.24, HLA-DR, tenascin, an angiogenesis factor, VEGF, PIGF,
ED-B fibronectin, an oncogene, an oncogene product, CD66a-d,
necrosis antigens, Ii, IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4) and
TRAIL-R2 (DR5).
[0353] D. Recombinant Receptors
[0354] Among the provided binding molecules, e.g., ROR1 binding
molecules, are cell surface proteins, such as recombinant
receptors, such as those that include one of the provided
antibodies or antigen-binding fragments. Also provided are
polynucleotides that encode all or a portion of such cell surface
proteins, e.g., receptors. The receptors include antigen receptors
and other chimeric receptors that specifically bind to ROR1, such
as receptors containing the provided anti-ROR1 antibodies, e.g.,
antigen-binding fragments. Among the antigen receptors are
functional non-TCR antigen receptors, such as chimeric antigen
receptors (CARs). The ROR1-binding receptors generally contain
antibodies (e.g., antigen-binding fragments), and/or other binding
peptides that specifically bind to ROR1, such as to ROR1 proteins,
such as a human ROR1 protein. Also provided are cells expressing
the recombinant receptors, compositions containing such cells and
uses thereof in adoptive cell therapy, such as treatment of
diseases and disorders associated with ROR1 expression,
compositions and articles of manufacture and uses of the same.
[0355] Among the provided polynucleotides are those that encode
recombinant receptors, such as antigen receptors, that specifically
bind ROR1. In some aspects, the encoded receptors, such as those
containing ROR1-binding polypeptides, and compositions and articles
of manufacture and uses of the same, also are provided. The
provided polynucleotides can be incorporated into constructs, such
as deoxyribonucleic acid (DNA) or RNA constructs, such as those
that can be introduced into cells for expression of the encoded
recombinant ROR1-binding receptors.
[0356] I. Exemplary Receptors
[0357] The provided ROR1-binding cell surface proteins, such as
receptors, generally contain an extracellular antigen-binding
domain and an intracellular signaling region. Among the provided
receptors, e.g., recombinant receptors, are polypeptides containing
the provided antibodies or antigen-binding fragment thereof, such
as one or more of the provided anti-ROR1 antibody or fragment
thereof. In some embodiments, the provided cell surface proteins
specifically bind to ROR1, such as a human ROR1.
[0358] Among the antigen receptors are chimeric and/or functional
non-TCR antigen receptors, such as chimeric antigen receptors
(CARs). The chimeric receptors, such as CARs, generally include an
extracellular antigen binding domain that includes, is, or is
comprises an anti-ROR1 antibody, such as an anti-ROR1 antibody or
fragment thereof described herein. In some embodiments, the
chimeric receptors, e.g., CARs, include an intracellular signaling
domain. In some embodiments, the chimeric receptors also include a
spacer and/or a transmembrane domain. In some embodiments, the
spacer is located between the extracellular antigen-binding domain
and the transmembrane domain. In some embodiments, the CAR contains
an extracellular antigen-binding domain, a spacer, a transmembrane
region and an intracellular signaling region. Exemplary CARs
provided herein include those containing an antigen-binding domain
comprising an antibody or antigen-binding fragment thereof
described herein, e.g., in Section I.A and/or in Table 2, or those
described in Section I.D herein, in Table 3 and/or in Table E1.
Also provided are CARs encoded by the polynucleotides described in
Section I.E herein, in Table 3 and/or in Table E1.
[0359] In some cases, CARs are referred to as first, second, and/or
third generation CARs. In some aspects, a first generation CAR is
one that solely provides a CD3-chain induced signal upon antigen
binding; in some aspects, a second-generation CARs is one that
provides such a signal and costimulatory signal, such as one
including an intracellular signaling domain from a costimulatory
receptor such as CD28 or CD137; in some aspects, a third generation
CAR in some aspects is one that includes multiple costimulatory
domains of different costimulatory receptors.
[0360] In some embodiments, the CAR contains an extracellular
antigen-binding domain, in some cases comprising an antibody, e.g.,
an antibody fragment that binds ROR1, a transmembrane domain that
is or contains a transmembrane portion of CD28 or a functional
variant thereof, and an intracellular signaling domain containing a
signaling portion of CD28 or functional variant thereof and a
signaling portion of CD3 zeta or functional variant thereof. In
some embodiments, the CAR contains an extracellular antigen-binding
domain, in some cases comprising an antibody, e.g., antibody
fragment that binds ROR1, a transmembrane domain that is or
contains a transmembrane portion of CD28 or a functional variant
thereof, and an intracellular signaling domain containing a
signaling portion of a 4-1BB or functional variant thereof and a
signaling portion of CD3 zeta or functional variant thereof. In
some such embodiments, the receptor further includes a spacer
containing a portion of an Ig molecule, such as a human Ig
molecule, such as an Ig hinge, e.g., an IgG4 hinge, such as a
hinge-only spacer.
[0361] In some embodiments, the chimeric antigen receptor includes
an extracellular portion containing the antibody or fragment
thereof described herein. In some aspects, the chimeric antigen
receptor includes an extracellular portion containing the antibody
or fragment thereof described herein and an intracellular signaling
domain. In some embodiments, the antibody or fragment thereof
includes an scFv and the intracellular signaling region contains an
ITAM. In some aspects, the intracellular signaling domain includes
a signaling domain of a zeta chain of a CD3-zeta (CD3.zeta.) chain.
In some embodiments, the chimeric antigen receptor includes a
transmembrane domain linking the extracellular domain and the
intracellular signaling domain.
[0362] Other exemplary antigen receptors, including CARs, and
methods for engineering and introducing such receptors into cells,
include those described, for example, in WO200014257, WO2013126726,
WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154,
WO2013/123061 U.S. patent application publication numbers
US2002131960, US2013287748, 0520130149337, U.S. Pat. Nos.
6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179,
6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353,
and 8,479,118, and European patent application number EP2537416,
and/or those described by Sadelain et al., Cancer Discov. 2013
April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338;
Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39;
Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the
antigen receptors include a CAR as described in U.S. Pat. No.
7,446,190, and those described in WO/2014055668 A1. Exemplary of
the CARs include CARs as disclosed in any of the aforementioned
publications, such as WO2014031687, U.S. Pat. Nos. 8,339,645,
7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, 8,389,282,
e.g., and in which the antigen-binding portion, e.g., scFv, is
replaced by an antibody, e.g., as provided herein.
[0363] Other ROR1-targeting CARs are described, for example, by
Hudecek et al., Clin Cancer Res, 19(12), 3153-3164 (2013) and
Baskar et al. MAbs. 4(3): 349-361 (2012). See also WO2014031687;
US2012/20058051.
[0364] a. Extracellular Antigen-Binding Domain
[0365] Among the chimeric receptors are chimeric antigen receptors
(CARs). The chimeric receptors, such as CARs, generally include an
extracellular antigen binding domain that includes, is, or is
comprised within, one or more of the provided anti-ROR1 antibodies
or antigen-binding fragments. Thus, the chimeric receptors, e.g.,
CARs, typically include in their extracellular portions one or more
ROR1-binding molecules, such as one or more antigen-binding
fragment, domain, or portion, or one or more antibody variable
domains and/or antibody molecules, such as those described herein.
In some embodiments, the CAR includes a ROR1-binding portion or
portions of the antibody molecule, such as a variable heavy
(V.sub.H) chain region and/or variable light (V.sub.L) chain region
of the antibody, e.g., an scFv. In some embodiments, the CAR
includes a ROR1-binding portion or portions of the antibody
molecule, such as a variable heavy (V.sub.H) chain region and a
variable light (V.sub.L) chain region of the antibody, e.g., an
scFv. In some aspects, the CAR includes one or more of any of the
ROR1-binding antibodies or antigen-binding fragments thereof
described herein, e.g., in Section I.A.
[0366] In some embodiments, Table 2 provides the SEQ ID NOS: of
exemplary antigen-binding domains, such as antibodies or
antigen-binding fragments, that can be comprised in the provided
ROR1-binding receptors, such as anti-ROR1 chimeric antigen
receptors (CARs). In some aspects, the CAR comprises an scFv
described in Table 2, for example, as a part of the extracellular
antigen-binding domain. In some aspects, the CAR comprises a
V.sub.H region described in Table 2, for example, as a part of the
extracellular antigen-binding domain. In some aspects, the CAR
comprises a V.sub.L region described in Table 2, for example, as a
part of the extracellular antigen-binding domain. In some
embodiments, the ROR1-binding receptor contains a ROR1-binding
antibody or fragment thereof, comprising a V.sub.H region that
comprises a CDR-H1, a CDR-H2 and a CDR-H3 sequence and a V.sub.L
region that comprises a CDR-L1, a CDR-L2 and a CDR-L3 sequence set
forth in the SEQ ID NOS: listed in each row of Table 2. In some
embodiments, the ROR1-binding receptor contains a ROR1-binding
antibody or fragment thereof, comprising a V.sub.H region sequence
and a V.sub.L region sequence set forth in the SEQ ID NOS: listed
in each row of Table 2, or an antibody comprising a V.sub.H and
V.sub.L region amino acid sequence that has at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
V.sub.H region sequence and the V.sub.L region sequence set forth
in the SEQ ID NOS: listed in each row of Table 2. In some
embodiments, the ROR1-binding receptor contains a ROR1-binding
antibody or fragment thereof, comprising a V.sub.H region sequence
and a V.sub.L region sequence set forth in the SEQ ID NOS: listed
in each row of Table 2. In some embodiments, the ROR1-binding
receptor contains a ROR1-binding antibody or fragment thereof,
comprising an scFv sequence set forth in the SEQ ID NOS: listed in
each row of Table 2, or an antibody comprising an scFv amino acid
sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the scFv sequence set forth in the
SEQ ID NOS: listed in each row of Table 2. In some embodiments, the
ROR1-binding receptor contains a ROR1-binding antibody or fragment
thereof, comprising an scFv sequence set forth in the SEQ ID NOS:
listed in each row of Table 2.
[0367] In some embodiments, the provided CARs can include an
extracellular antigen-binding domain that contains all or a portion
of an antibody, including polyclonal and monoclonal antibodies,
including intact antibodies and functional (antigen-binding)
antibody fragments, including fragment antigen binding (Fab)
fragments, F(ab')2 fragments, Fab' fragments, Fv fragments,
recombinant IgG (rIgG) fragments, heavy chain variable (V.sub.H)
regions capable of specifically binding the antigen, single chain
antibody fragments, including single chain variable fragments
(scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody)
fragments. In some aspects, the antibody or fragment thereof
contained in the CARs include genetically engineered and/or
otherwise modified forms of immunoglobulins, such as intrabodies,
peptibodies, chimeric antibodies, fully human antibodies, humanized
antibodies, and heteroconjugate antibodies, multispecific, e.g.,
bispecific or trispecific, antibodies, diabodies, triabodies, and
tetrabodies, tandem di-scFv, tandem tri-scFv.
[0368] b. Spacer
[0369] In some embodiments, the recombinant receptor such as the
CAR, such as the antibody portion thereof, further includes a
spacer (in some cases also called a spacer region), which may be or
include at least a portion of an immunoglobulin constant region or
variant or modified version thereof, such as a hinge region, e.g.,
an IgG4 hinge region, and/or a C.sub.H1/C.sub.L, C.sub.H2 and/or
C.sub.H3 and/or Fc region. In some embodiments, the constant region
or portion is of a human IgG, such as IgG4, IgG2 or IgG1. In some
aspects, the portion of the constant region serves as a spacer
between the antigen-recognition component, e.g., scFv, and
transmembrane domain.
[0370] In some embodiments, the length of the spacer is adjusted to
optimize the biophysical synapse distance between the
CAR-expressing cell, such as a CAR-expressing cell, and the target
of the CAR, such as a ROR1-expressing tumor cell. In some
embodiments, the CAR is expressed by a T cell, and the length of
the spacer is adjusted to a length that is compatible for T cell
activation or to optimize CAR T-cell performance.
[0371] In some embodiments, the spacer can be of a length that
provides for increased responsiveness of the cell following antigen
binding, as compared to in the absence of the spacer or as compared
to an alternative spacer of a different length (e.g. longer in
length). In some examples, the spacer is at or about 12 amino acids
in length or is no more than at or about 12 amino acids in length.
In some examples, the spacer is at or about 15 amino acids in
length or is no more than at or about 15 amino acids in length.
[0372] Exemplary spacers include those having at least at or about
10 to at or about 300 amino acids, at or about 10 to at or about
229 amino acids, at or about 10 to at or about 200 amino acids, at
or about 10 to at or about 175 amino acids, at or about 10 to at or
about 150 amino acids, at or about 10 to at or about 125 amino
acids, at or about 10 to at or about 100 amino acids, at or about
10 to at or about 75 amino acids, at or about 10 to at or about 50
amino acids, at or about 10 to at or about 40 amino acids, at or
about 10 to at or about 30 amino acids, at or about 10 to at or
about 20 amino acids, or at or about 10 to at or about 15 amino
acids in length, and including any integer between the endpoints of
any of the listed ranges. Exemplary spacers include those having at
least at or about at or about 50 to at or about 175 amino acids, at
or about 50 to at or about 150 amino acids, at or about 10 to at or
about 125 amino acids, at or about 50 to at or about 100 amino
acids, at or about 100 to at or about 300 amino acids, at or about
100 to at or about 250 amino acids, at or about 125 to at or about
250 amino acids, or at or about 200 to at or about 250 amino acids,
and including any integer between the endpoints of any of the
listed ranges. In some embodiments, a spacer is at least at or
about 12 amino acids, at least at or about 119 amino acids, at
least at or about 125 amino acids, at least at or about 200 amino
acids, or at least at or about 220 amino acids, or at least at or
about 225 amino acids in length. In some embodiments, a spacer is
at least at or about 13 amino acids, at least at or about 120 amino
acids, at least at or about 125 amino acids, at least at or about
200 amino acids, or at least at or about 220 amino acids, or at
least at or about 229 amino acids in length. In some embodiments, a
spacer is at or about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25 amino acids or less in length. In some embodiments,
the spacer is at least at or about 100 amino acids in length, such
as at least at or about 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 121, 122, 123, 124, 125, 130, 135, 140, 145, 150,
160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids in
length.
[0373] In some embodiments, the spacer is at least at or about 125
to at or about 300 amino acids, at or about 125 to at or about 250
amino acids, at or about 125 to at or about 230 amino acids, at or
about 125 to at or about 200 amino acids, at or about 125 to at or
about 180 amino acids, at or about 125 to at or about 150 amino
acids, at or about 150 to at or about 300 amino acids, at or about
150 to at or about 250 amino acids, at or about 150 to at or about
230 amino acids, at or about 150 to at or about 200 amino acids, at
or about 150 to at or about 180 amino acids, at or about 180 to at
or about 300 amino acids, at or about 180 to at or about 250 amino
acids, at or about 180 to at or about 230 amino acids, at or about
180 to at or about 200 amino acids, at or about 200 to at or about
300 amino acids, at or about 200 to at or about 250 amino acids, at
or about 200 to at or about 230 amino acids, at or about 230 to at
or about 300 amino acids, at or about 230 to at or about 250 amino
acids in length or 250 to at or about 300 amino acids in length. In
some embodiments, the spacer is at least at or about 129, 130, 140,
150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229 or 230 amino acids in length, or a length
between any of the foregoing.
[0374] Exemplary spacers include an IgG hinge alone, an IgG hinge
linked to one or more of a C.sub.H2 and C.sub.H3 domain, or IgG
hinge linked to the C.sub.H3 domain. In some embodiments, the
spacer includes an IgG hinge alone. In some embodiments, the IgG
hinge, C.sub.H2 and/or C.sub.H3 can be derived all or in part from
IgG4 or IgG2, such as all or in part from human IgG4 or human IgG2.
In some embodiments, the spacer can be a chimeric polypeptide
containing one or more of a hinge, C.sub.H2 and/or C.sub.H3
sequence(s) derived from IgG4, IgG2, and/or IgG2 and IgG4. In some
embodiments, the hinge region comprises all or a portion of an IgG4
hinge region. In some embodiments, the hinge region comprises all
or a portion of an IgG4 hinge region and/or of an IgG2 hinge
region, wherein the IgG4 hinge region is optionally a human IgG4
hinge region and the IgG2 hinge region is optionally a human IgG2
hinge region; the C.sub.H2 region comprises all or a portion of an
IgG4 C.sub.H2 region and/or of an IgG2 C.sub.H2 region, wherein the
IgG4 C.sub.H2 region is optionally a human IgG4 C.sub.H2 region and
the IgG2 C.sub.H2 region is optionally a human IgG2 C.sub.H2
region; and/or the C.sub.H3 region comprises all or a portion of an
IgG4 C.sub.H3 region and/or of an IgG2 C.sub.H3 region, wherein the
IgG4 C.sub.H3 region is optionally a human IgG4 C.sub.H3 region and
the IgG2 C.sub.H3 region is optionally a human IgG2 C.sub.H3
region. In some embodiments, the hinge, C.sub.H2 and C.sub.H3
comprises all or a portion of each of a hinge region, C.sub.H2 and
C.sub.H3 from IgG4. In some embodiments, the hinge region is
chimeric and comprises a hinge region from human IgG4 and human
IgG2; the C.sub.H2 region is chimeric and comprises a C.sub.H2
region from human IgG4 and human IgG2; and/or the C.sub.H3 region
is chimeric and comprises a C.sub.H3 region from human IgG4 and
human IgG2. In some embodiments, the spacer comprises an IgG4/2
chimeric hinge or a modified IgG4 hinge comprising at least one
amino acid replacement compared to human IgG4 hinge region; an
human IgG2/4 chimeric C.sub.H2 region; and a human IgG4 C.sub.H3
region.
[0375] In some embodiments, the spacer comprises or consists of all
or a portion of an immunoglobulin hinge or a modified version
thereof. In some embodiments, the spacer is at or about 15 amino
acids or less in length. In some embodiments, the spacer comprises
or consists of all or a portion of an immunoglobulin hinge,
optionally an IgG4 hinge, or a modified version thereof and/or
comprises about 15 amino acids or less. In some embodiments, the
spacer is at or about 13 amino acids in length and/or comprises or
consists of all or a portion of an immunoglobulin hinge, optionally
an IgG4, or a modified version thereof. In some embodiments, the
spacer is at or about 12 amino acids in length and/or comprises or
consists of all or a portion of an immunoglobulin hinge, optionally
an IgG4, or a modified version thereof. In some embodiments, the
spacer comprises or consists of the sequence of SEQ ID NO: 1, 26,
27, 28, 29, 31, 32, 33 or 135, or a variant of any of the foregoing
having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some
embodiments, the spacer comprises the formula X.sub.1PPX.sub.2P
(SEQ ID NO:25), where X.sub.1 is glycine, cysteine or arginine and
X.sub.2 is cysteine or threonine. In some embodiments, the spacer
does not comprise a CD28 extracellular region or a CD8
extracellular region. In certain cases, the spacer has a methionine
residue at the C-terminus.
[0376] In some of any such embodiments, the spacer is or contains
the sequence set forth in SEQ ID NO:1. In some of any such
embodiments, the spacer is or contains the amino acid sequence
encoded by SEQ ID NO: 2 or 30 or a nucleic acid sequence having at
least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO: 2 or 30. In some of any such embodiments, the spacer is or
contains the amino acid sequence encoded by SEQ ID NO: 30.
[0377] In some of any such embodiments, the spacer is or contains
the sequence set forth in SEQ ID NO:135. In some of any such
embodiments, the spacer is or contains the amino acid sequence
encoded by SEQ ID NO: 192 or 136 or a nucleic acid sequence having
at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID NO: 192 or 136. In some of any such embodiments, the spacer
is or contains the amino acid sequence encoded by SEQ ID NO:
136.
[0378] In some embodiments, the spacer is or comprises IgG hinge
linked to the C.sub.H3 domain, e.g., of a human immunoglobulin,
such as IgG4 and/or IgG2. In some aspects, the spacer is at or
about 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,
123, 124 or 125 amino acids in length, or has a length between any
of the foregoing. In some aspects, the spacer is at or about 119 or
120 amino acids in length. In some embodiments, the spacer
comprises or consists of the sequence of SEQ ID NO: 3 or 138, or a
variant of any of the foregoing having at least 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity thereto.
[0379] In some of any such embodiments, the spacer is or contains
an amino acid sequence having at least at or about 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 138. In some of any such embodiments, the
spacer is or contains the sequence set forth in SEQ ID NO:138. In
some of any such embodiments, the spacer is or contains the amino
acid sequence encoded by SEQ ID NO: 193 or 139 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 193 or 139. In some of any such
embodiments, the spacer is or contains the amino acid sequence
encoded by SEQ ID NO: 139.
[0380] In some of any such embodiments, the spacer is at or about
120 amino acids in length. In some of any such embodiments, the
spacer is or contains an amino acid sequence having at least at or
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity to SEQ ID NO: 3. In some of any such
embodiments, the spacer is or contains the sequence set forth in
SEQ ID NO:3. In some of any such embodiments, the spacer is or
contains the amino acid sequence encoded by SEQ ID NO: 4 or 137 or
a nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 4 or 137. In some of
any such embodiments, the spacer is or contains the amino acid
sequence encoded by SEQ ID NO: 137.
[0381] In some embodiments, the spacer can be from all or in part
from IgG4 and/or IgG2 and can contain mutations, such as one or
more single amino acid mutations in one or more domains. In some
examples, the amino acid modification is a substitution of a
proline (P) for a serine (S) in the hinge region of an IgG4. In
some embodiments, the amino acid modification is a substitution of
a glutamine (Q) for an asparagine (N) to reduce glycosylation
heterogeneity, such as an N177Q mutation at position 177, in the
C.sub.H2 region, of the full-length IgG4 Fc sequence set forth in
SEQ ID NO: 48 or an N176Q at position 176, in the C.sub.H2 region,
of the full-length IgG2 Fc sequence set forth in SEQ ID NO:49. In
some embodiments, the spacer is or comprises an IgG4/2 chimeric
hinge or a modified IgG4 hinge; an IgG2/4 chimeric C.sub.H2 region;
and an IgG4 C.sub.H3 region and optionally is about 228 or 229
amino acids in length; or a spacer set forth in SEQ ID NO: 37 or
194.
[0382] In some of any such embodiments, the spacer is or contains
an amino acid sequence having at least at or about 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO:194. In some of any such embodiments, the
spacer is or contains the sequence set forth in SEQ ID NO: 194. In
some of any such embodiments, the spacer is or contains the amino
acid sequence encoded by SEQ ID NO: 195 or 196 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 195 or 196. In some of any such
embodiments, the spacer is or contains the amino acid sequence
encoded by SEQ ID NO: 196.
[0383] In some of any such embodiments, the spacer is or contains
an amino acid sequence having at least at or about 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO:37. In some of any such embodiments, the
spacer is or contains the sequence set forth in SEQ ID NO: 37. In
some of any such embodiments, the spacer is or contains the amino
acid sequence encoded by SEQ ID NO: 38 or 140 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 38 or 140. In some of any such
embodiments, the spacer is or contains the amino acid sequence
encoded by SEQ ID NO: 140.
[0384] In some embodiments, the spacer is encoded by a
polynucleotide that has been optimized for codon expression and/or
to eliminate splice sites such as cryptic splice sites. In some
embodiments, the coding sequence for the spacer comprises the
nucleic acid sequence set forth in SEQ ID NO: 30. In some
embodiments, the coding sequence for the spacer comprises the
nucleic acid sequence set forth in SEQ ID NO: 136. In some
embodiments, the coding sequence for the spacer comprises the
nucleic acid sequence set forth in SEQ ID NO: 137. In some
embodiments, the coding sequence for the spacer comprises the
nucleic acid sequence set forth in SEQ ID NO: 139. In some
embodiments, the coding sequence for the spacer comprises the
nucleic acid sequence set forth in SEQ ID NO: 140. In some
embodiments, the coding sequence for the spacer comprises the
nucleic acid sequence set forth in SEQ ID NO: 196.
[0385] Additional exemplary spacers include, but are not limited
to, those described in Hudecek et al. (2013) Clin. Cancer Res.,
19:3153, Hudecek et al. (2015) Cancer Immunol. Res., 3(2):125-135,
or WO2014031687. In some embodiments, the nucleotide sequence of
the spacer is optimized to reduce RNA heterogeneity upon
expression. In some embodiments, the nucleotide sequence of the
spacer is optimized to reduce cryptic splice sites or reduce the
likelihood of a splice event at a splice site.
[0386] In some embodiments, the spacer has the amino acid sequence
set forth in SEQ ID NO:1, and is encoded by the polynucleotide
sequence set forth in SEQ ID NO:2. In some embodiments, the spacer
has the amino acid sequence set forth in SEQ ID NO:135, and is
encoded by the polynucleotide sequence set forth in SEQ ID NO:192.
In some embodiments, the spacer has the amino acid sequence set
forth in SEQ ID NO:3, and is encoded by the polynucleotide sequence
set forth in SEQ ID NO:4. In some embodiments, the spacer has the
amino acid sequence set forth in SEQ ID NO:138, and is encoded by
the polynucleotide sequence set forth in SEQ ID NO:193. In some
embodiments, the spacer has the amino acid sequence set forth in
SEQ ID NO:37, and is encoded by the polynucleotide sequence set
forth in SEQ ID NO:38. In some embodiments, the spacer has the
amino acid sequence set forth in SEQ ID NO:194, and is encoded by
the polynucleotide sequence set forth in SEQ ID NO:195.
[0387] In some embodiments, the spacer is encoded by a
polynucleotide that has been optionally optimized for codon usage
and/or to reduce RNA heterogeneity, e.g., by removing cryptic
splice sites. In some embodiments, the spacer has the amino acid
sequence set forth in SEQ ID NO:1, and is encoded by the
polynucleotide sequence set forth in SEQ ID NO:30. In some
embodiments, the spacer has the amino acid sequence set forth in
SEQ ID NO:135, and is encoded by the polynucleotide sequence set
forth in SEQ ID NO:136. In some embodiments, the spacer has the
amino acid sequence set forth in SEQ ID NO:3, and is encoded by the
polynucleotide sequence set forth in SEQ ID NO:137. In some
embodiments, the spacer has the amino acid sequence set forth in
SEQ ID NO:138, and is encoded by the polynucleotide sequence set
forth in SEQ ID NO:139. In some embodiments, the spacer has the
amino acid sequence set forth in SEQ ID NO:37, and is encoded by
the polynucleotide sequence set forth in SEQ ID NO:140. In some
embodiments, the spacer has the amino acid sequence set forth in
SEQ ID NO:194, and is encoded by the polynucleotide sequence set
forth in SEQ ID NO:196. In some embodiments, the spacer has an
amino acid sequence that exhibits at least at or about 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
sequence identity to SEQ ID NO:1, 3 or 37 and is encoded by a
polynucleotide that has been optionally optimized for codon usage
and/or to reduce RNA heterogeneity. In some embodiments, the spacer
has an amino acid sequence that exhibits at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% sequence identity to SEQ ID NO:135, 138 or 194 and is encoded
by a polynucleotide that has been optionally optimized for codon
usage and/or to reduce RNA heterogeneity.
[0388] c. Transmembrane Domain
[0389] The antigen-recognition component generally is linked to one
or more intracellular signaling components, such as signaling
components that mimic activation through an antigen receptor
complex, such as a TCR complex, in the case of a CAR, and/or signal
via another cell surface receptor. Thus, in some embodiments, a
ROR1-binding molecule (e.g., antibody or antigen binding fragment
thereof) is linked to one or more transmembrane domains such as
those described herein and intracellular signaling domains
comprising one or more intracellular components such as those
described herein. In some embodiments, the transmembrane domain is
fused to the extracellular domain. In one embodiment, a
transmembrane domain that naturally is associated with one of the
domains in the receptor, e.g., CAR, is used. In some instances, the
transmembrane domain is selected or modified by amino acid
substitution to avoid binding of such domains to the transmembrane
domains of the same or different surface membrane proteins to
minimize interactions with other members of the receptor
complex.
[0390] The transmembrane domain in some embodiments is derived
either from a natural or from a synthetic source. Where the source
is natural, the domain in some aspects is derived from any
membrane-bound or transmembrane protein. Transmembrane domains
include those derived from (i.e. comprise at least the
transmembrane domain(s) of) the alpha, beta or zeta chain of the
T-cell receptor, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28,
CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, and/or CD154. For
example, the transmembrane domain can be a CD28 transmembrane
domain that comprises the sequence of amino acids set forth in SEQ
ID NO: 149, encoded by the nucleic acid sequence set forth in SEQ
ID NO:147 or 148. For example, the transmembrane domain can be a
CD28 transmembrane domain that comprises the sequence of amino
acids set forth in SEQ ID NO: 8, encoded by the nucleic acid
sequence set forth in SEQ ID NO:197 or 198. In some embodiments,
the transmembrane domain of the receptor, e.g., the CAR is a
transmembrane domain of human CD28 or variant thereof, e.g., a
27-amino acid transmembrane domain of a human CD28 (Accession No.:
P10747.1), or a 28-amino acid sequence, or is a transmembrane
domain that comprises the sequence of amino acids set forth in SEQ
ID NO:8 or 149 or a sequence of amino acids that exhibits at least
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more sequence identity to SEQ ID NO: 8 or 149. In some
embodiments, the transmembrane domain is encoded by a
polynucleotide that has been optionally optimized for codon usage
and/or to reduce RNA heterogeneity, e.g., by removing cryptic
splice sites. In some embodiments, the transmembrane domain has the
amino acid sequence set forth in SEQ ID NO:8 or 149, and is encoded
by the polynucleotide sequence set forth in SEQ ID NO:147, 148, 197
or 198, or a polynucleotide sequence having at least 90% sequence
identity thereto. In certain cases, the transmembrane domain has a
methionine residue at the N-terminus.
[0391] In some of any such embodiments, the transmembrane domain is
or contains SEQ ID NO: 8 or an amino acid sequence having at least
at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
sequence identity to SEQ ID NO: 8. In some of any such embodiments,
the transmembrane domain is or contains the sequence set forth in
SEQ ID NO: 8. In some of any such embodiments, the transmembrane
domain is or contains the amino acid sequence encoded by SEQ ID NO:
197 or 198 or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 197 or
198. In some of any such embodiments, the transmembrane domain is
or contains the amino acid sequence encoded by SEQ ID NO: 198.
[0392] In some of any such embodiments, the transmembrane domain is
or contains SEQ ID NO: 149 or an amino acid sequence having at
least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% sequence identity to SEQ ID NO: 149. In some of any such
embodiments, the transmembrane domain is or contains the sequence
set forth in SEQ ID NO: 149. In some of any such embodiments, the
transmembrane domain is or contains the amino acid sequence encoded
by SEQ ID NO: 147 or 148 or a nucleic acid sequence having at least
at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 147 or 148. In some of any such embodiments, the transmembrane
domain is or contains the amino acid sequence encoded by SEQ ID NO:
148.
[0393] Alternatively, the transmembrane domain in some embodiments
is synthetic. In some aspects, the synthetic transmembrane domain
comprises predominantly hydrophobic residues such as leucine and
valine. In some aspects, a triplet of phenylalanine, tryptophan and
valine will be found at each end of a synthetic transmembrane
domain. In some embodiments, the linkage is by linkers, spacers,
and/or transmembrane domain(s).
[0394] d. Intracellular Signaling Components
[0395] Among the intracellular signaling domains are those that
mimic or approximate a signal through a natural antigen receptor, a
signal through such a receptor in combination with a costimulatory
receptor, and/or a signal through a costimulatory receptor alone.
In some embodiments, a short oligo- or polypeptide linker, for
example, a linker of between 2 and 10 amino acids in length, such
as one containing glycines and serines, e.g., glycine-serine
doublet, is present and forms a linkage between the transmembrane
domain and the intracellular signaling domain of the CAR.
[0396] The receptor, e.g., the CAR, generally includes an
intracellular signaling region comprising at least one
intracellular signaling component or components. In some
embodiments, the receptor includes an intracellular component or
signaling domain of a TCR complex, such as a TCR CD3 chain that
mediates T-cell activation and cytotoxicity, e.g., CD3 zeta
(CD3-.zeta.) chain. Thus, in some aspects, the ROR1-binding
antibody is linked to one or more cell signaling modules. In some
embodiments, cell signaling modules include CD3 transmembrane
domain, CD3 intracellular signaling domains, and/or other CD
transmembrane domains. In some embodiments, the receptor, e.g.,
CAR, further includes a portion of one or more additional molecules
such as Fc receptor .gamma., CD8, CD4, CD25, or CD16. For example,
in some aspects, the CAR includes a chimeric molecule between
CD3-zeta (CD3-.zeta.) or Fc receptor .gamma. and CD8, CD4, CD25 or
CD16.
[0397] In some embodiments, upon ligation of the CAR, the
cytoplasmic domain or intracellular signaling region of the CAR
stimulates and/or activates at least one of the normal effector
functions or responses of the immune cell, e.g., T cell engineered
to express the CAR. For example, in some contexts, the CAR induces
a function of a T cell such as cytolytic activity or T-helper
activity, such as secretion of cytokines or other factors. In some
embodiments, a truncated portion of an intracellular signaling
domain of an antigen receptor component or costimulatory molecule
is used in place of an intact immunostimulatory chain, for example,
if it transduces the effector function signal. In some embodiments,
the intracellular signaling domain or domains include the
cytoplasmic sequences of the T cell receptor (TCR), and in some
aspects also those of co-receptors that in the natural context act
in concert with such receptor to initiate signal transduction
following antigen receptor engagement, and/or any derivative or
variant of such molecules, and/or any synthetic sequence that has
the same functional capability.
[0398] In the context of a natural TCR, full activation generally
requires not only signaling through the TCR, but also a
costimulatory signal. Thus, in some embodiments, to promote full
activation, a component for generating secondary or co-stimulatory
signal is also included in the CAR. In other embodiments, the CAR
does not include a component for generating a costimulatory signal.
In some aspects, an additional CAR is expressed in the same cell
and provides the component for generating the secondary or
costimulatory signal.
[0399] T cell activation is in some aspects described as being
mediated by two classes of cytoplasmic signaling sequences: those
that initiate antigen-dependent primary activation through the TCR
(primary cytoplasmic signaling sequences), and those that act in an
antigen-independent manner to provide a secondary or co-stimulatory
signal (secondary cytoplasmic signaling sequences). In some
aspects, the CAR includes one or both of such classes of
cytoplasmic signaling sequences.
[0400] In some aspects, the CAR includes a primary cytoplasmic
signaling sequence that regulates primary stimulation and/or
activation of the TCR complex. Primary cytoplasmic signaling
sequences that act in a stimulatory manner may contain signaling
motifs which are known as immunoreceptor tyrosine-based activation
motifs or ITAMs. Examples of ITAM containing primary cytoplasmic
signaling sequences include those derived from TCR or CD3 zeta, FcR
gamma, CD3 gamma, CD3 delta and CD3 epsilon. In some embodiments,
the intracellular signaling region in the CAR contain(s) a
cytoplasmic signaling domain, portion thereof, or sequence derived
from CD3 zeta. In some embodiments the CD3 zeta comprises the
sequence of amino acids set forth in SEQ ID NO:13, 14 or 15,
encoded by the nucleic acid sequence set forth in SEQ ID NO: 150 or
182. In some embodiments, the CD3 zeta is encoded by a
polynucleotide that has been optionally optimized for codon usage
and/or to reduce RNA heterogeneity, e.g., by removing cryptic
splice sites. In some embodiments, the CD3 zeta has the amino acid
sequence set forth in SEQ ID NO:13, and is encoded by the
polynucleotide sequence set forth in SEQ ID NO:150. In some
embodiments, the intracellular signaling domain comprises a human
CD3 zeta stimulatory signaling domain or functional variant
thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human
CD3 (Accession No.: P20963.2) or a CD3 zeta signaling domain as
described in U.S. Pat. Nos. 7,446,190 or 8,911,993. In some
embodiments, the intracellular signaling domain comprises the
sequence of amino acids set forth in SEQ ID NO: 13, 14 or 15 or a
sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity to SEQ ID NO: 13, 14 or 15.
[0401] In some embodiments, the CAR includes a signaling domain
(e.g., an intracellular or cytoplasmic signaling domain) and/or
transmembrane portion of a costimulatory molecule, such as a T cell
costimulatory molecule. Exemplary costimulatory molecules include
CD28, 4-1BB, OX40, DAP10, and ICOS. For example, a costimulatory
molecule can be derived from 4-1BB and can comprise the amino acid
sequence set forth in SEQ ID NO: 12, encoded by the nucleotide
sequence set forth in SEQ ID NO: 154 or 155. In some embodiments,
the costimulatory molecule from 4-1BB is encoded by a
polynucleotide that has been optionally optimized for codon usage
and/or to reduce RNA heterogeneity, e.g., by removing cryptic
splice sites. In some embodiments, the costimulatory molecule from
4-1BB has the amino acid sequence set forth in SEQ ID NO:12, and is
encoded by the polynucleotide sequence set forth in SEQ ID NO:155.
In some embodiments, the intracellular domain comprises an
intracellular costimulatory signaling domain of 4-1BB or functional
variant or portion thereof, such as a 42-amino acid cytoplasmic
domain of a human 4-1BB (Accession No. Q07011.1) or functional
variant or portion thereof, such as the sequence of amino acids set
forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits
at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12. In
some embodiments, a costimulatory molecule can be derived from CD28
and can comprise the amino acid sequence set forth in SEQ ID NO:
10, encoded by the nucleotide sequence set forth in SEQ ID NO: 183.
In some embodiments, the intracellular signaling domain comprises
an intracellular costimulatory signaling domain of human CD28 or
functional variant or portion thereof, such as a 41 amino acid
domain thereof and/or such a domain with an LL to GG substitution
at positions 186-187 of a native CD28 protein. In some embodiments,
the intracellular signaling domain can comprise the sequence of
amino acids set forth in SEQ ID NO: 10 or 11 or a sequence of amino
acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to
SEQ ID NO: 10 or 11. In some aspects, the same CAR includes both
the stimulatory or activating components (e.g., cytoplasmic
signaling sequence) and costimulatory components.
[0402] In some aspects, the transmembrane domain contains a
transmembrane portion of CD28. The extracellular domain and
transmembrane can be linked directly or indirectly. In some
embodiments, the extracellular domain and transmembrane are linked
by a spacer, such as any described herein. In some embodiments, the
chimeric antigen receptor contains an intracellular domain of a T
cell costimulatory molecule, such as between the transmembrane
domain and intracellular signaling domain. In some aspects, the T
cell costimulatory molecule is CD28 or 4-1BB.
[0403] In some embodiments, the stimulatory or activating
components are included within one CAR, whereas the costimulatory
component is provided by another CAR recognizing another antigen.
In some embodiments, the CARs include activating or stimulatory
CARs, and costimulatory CARs, both expressed on the same cell (see
WO 2014/055668). In some aspects, the ROR1-targeting CAR is the
stimulatory or activating CAR; in other aspects, it is the
costimulatory CAR. In some embodiments, the cells further include
inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine,
5(215) (December, 2013), such as a CAR recognizing an antigen other
than ROR1, whereby a stimulatory or an activating signal delivered
through the ROR1-targeting CAR is diminished or inhibited by
binding of the inhibitory CAR to its ligand, e.g., to reduce
off-target effects.
[0404] In some embodiments, the two receptors induce, respectively,
an activating and an inhibitory signal to the cell, such that
ligation of one of the receptor to its antigen activates the cell
or induces a response, but ligation of the second inhibitory
receptor to its antigen induces a signal that suppresses or dampens
that response. Examples are combinations of activating CARs and
inhibitory CARs (iCARs). Such a strategy may be used, for example,
to reduce the likelihood of off-target effects in the context in
which the activating CAR binds an antigen expressed in a disease or
condition but which is also expressed on normal cells, and the
inhibitory receptor binds to a separate antigen which is expressed
on the normal cells but not cells of the disease or condition.
[0405] In some aspects, the chimeric receptor is or includes an
inhibitory CAR (e.g. iCAR) and includes intracellular components
that dampen or suppress an immune response, such as an ITAM- and/or
co stimulatory-promoted response in the cell. Exemplary of such
intracellular signaling components are those found on immune
checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R,
TIM-3, TIGIT, LAIR-1, PGE2 receptors, EP2/4 Adenosine receptors
including A2AR. In some aspects, the engineered cell includes an
inhibitory CAR including a signaling domain of or derived from such
an inhibitory molecule, such that it serves to dampen the response
of the cell, for example, that induced by an activating and/or
costimulatory CAR.
[0406] In certain embodiments, the intracellular signaling region
comprises a CD28 transmembrane and signaling domain linked to a CD3
(e.g., CD3-zeta) intracellular domain. In some embodiments, the
intracellular signaling domain comprises a chimeric CD28 and 4-1BB
(CD137; TNFRSF9) co-stimulatory domains, linked to a CD3 zeta
intracellular domain.
[0407] In some embodiments, the CAR encompasses one or more, e.g.,
two or more, costimulatory domains and a stimulatory or an
activation domain, e.g., primary activation domain, in the
cytoplasmic portion. Exemplary CARs include intracellular
components of CD3-zeta, CD28, and 4-1BB.
[0408] In some embodiments, provided embodiments of anti-ROR1 CAR
contains an extracellular antigen-binding domain containing any of
the anti-ROR1 antibody or antigen-binding fragments described
herein, such as in Section I.A.1 and/or Table 2; a spacer
comprising an IgG4/2 chimeric hinge or a modified IgG4 hinge, such
as one that is about 12 amino acids in length, or a spacer set
forth in SEQ ID NO:1, such as encoded by the nucleotide sequence
set forth in SEQ ID NOS: 2 or 30; a transmembrane domain, such as a
transmembrane domain from a human CD28; and an intracellular
signaling region comprising a cytoplasmic signaling domain of a
CD3-zeta (CD3.zeta.) chain and a costimulatory signaling region. In
some embodiments, provided embodiments of anti-ROR1 CAR contains an
extracellular antigen-binding domain containing any of the
anti-ROR1 antibody or antigen-binding fragments described herein,
such as in Section I.A.1 and/or Table 2; a spacer comprising an
IgG4/2 chimeric hinge or a modified IgG4 hinge, such as one that is
about 13 amino acids in length, or a spacer set forth in SEQ ID
NO:135, such as encoded by the nucleotide sequence set forth in SEQ
ID NOS: 136 or 192; a transmembrane domain, such as a transmembrane
domain from a human CD28; and an intracellular signaling region
comprising a cytoplasmic signaling domain of a CD3-zeta (CD3.zeta.)
chain and a costimulatory signaling region.
[0409] In some embodiments, provided embodiments of anti-ROR1 CAR
contains an extracellular antigen-binding domain containing any of
the anti-ROR1 antibody or antigen-binding fragments described
herein, such as in Section I.A.1 and/or Table 2; a spacer
comprising a modified IgG4 hinge-C.sub.H3, such as one that is
about 119 amino acids in length, or a spacer set forth in SEQ ID
NO:3, such as encoded by the nucleotide sequence set forth in SEQ
ID NO:4 or 137; a transmembrane domain, such as a transmembrane
domain from a human CD28; and an intracellular signaling region
comprising a cytoplasmic signaling domain of a CD3-zeta (CD3.zeta.)
chain and a costimulatory signaling region. In some embodiments,
provided embodiments of anti-ROR1 CAR contains an extracellular
antigen-binding domain containing any of the anti-ROR1 antibody or
antigen-binding fragments described herein, such as in Section
I.A.1 and/or Table 2; a spacer comprising a modified IgG4
hinge-C.sub.H3, such as one that is about 120 amino acids in
length, or a spacer set forth in SEQ ID NO:138, such as encoded by
the nucleotide sequence set forth in SEQ ID NO:193 or 139; a
transmembrane domain, such as a transmembrane domain from a human
CD28; and an intracellular signaling region comprising a
cytoplasmic signaling domain of a CD3-zeta (CD3.zeta.) chain and a
costimulatory signaling region.
[0410] In some of any such embodiments, the transmembrane domain is
or comprises the sequence set forth in SEQ ID NO:8. In some of any
such embodiments, the transmembrane domain is or comprises the
sequence set forth in SEQ ID NO:149. In some of any such
embodiments, the costimulatory signaling region is an intracellular
signaling domain of human CD28, human 4-1BB or human ICOS or a
signaling portion thereof. In particular embodiments, the
intracellular signaling domain is an intracellular signaling domain
of human 4-1BB. In some of any such embodiments, the intracellular
signaling domain is or comprises the sequence set forth in SEQ ID
NO:12. In some of any such embodiments, the cytoplasmic signaling
domain is a human CD3-zeta cytoplasmic signaling domain, such as
set forth in SEQ ID NO:13. In some of any such embodiments, the
intracellular signaling region comprises the sequences set forth in
SEQ ID NO:13 and SEQ ID NO:12. In some of any such embodiments,
those described in Section I.D herein, in Table 3 and/or in Table
E1. Also provided are CARs encoded by the polynucleotides described
in Section I.E herein, in Table 3 and/or in Table E1. Also provided
are polynucleotides that contain any of the nucleotide sequences
described herein, e.g., encoding all of a portion of the provided
binding molecules. In certain cases, the transmembrane domain has a
methionine residue at the N-terminus. In certain cases, the spacer
has a methionine residue at the C-terminus.
[0411] In some of any of the provided embodiments, the anti-ROR1
chimeric antigen receptor is or comprises the sequence set forth in
SEQ ID NO: 184, 185, 186, 187, 188 or 189 or a sequence that
exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set
forth in SEQ ID NO: 184, 185, 186, 187, 188 or 189. In some of any
embodiments, the anti-ROR1 CARs has the amino acid sequence set
forth in SEQ ID NO:184, or an amino acid sequence that is at least
at or about 85%, at or about 86%, at or about 87%, at or about 88%,
at or about 89%, at or about 90%, at or about 91%, at or about 92%,
at or about 93%, at or about 94%, at or about 95%, at or about 96%,
at or about 97%, at or about 98% or at or about 99% sequence
identity to the amino acid sequence set forth in SEQ ID NO:184. In
some of any embodiments, the anti-ROR1 CARs has the amino acid
sequence set forth in SEQ ID NO:185, or an amino acid sequence that
is at least at or about 85%, at or about 86%, at or about 87%, at
or about 88%, at or about 89%, at or about 90%, at or about 91%, at
or about 92%, at or about 93%, at or about 94%, at or about 95%, at
or about 96%, at or about 97%, at or about 98% or at or about 99%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:185. In some of any embodiments, the anti-ROR1 CARs has the
amino acid sequence set forth in SEQ ID NO:186, or an amino acid
sequence that is at least at or about 85%, at or about 86%, at or
about 87%, at or about 88%, at or about 89%, at or about 90%, at or
about 91%, at or about 92%, at or about 93%, at or about 94%, at or
about 95%, at or about 96%, at or about 97%, at or about 98% or at
or about 99% sequence identity to the amino acid sequence set forth
in SEQ ID NO:186. In some of any embodiments, the anti-ROR1 CARs
has the amino acid sequence set forth in SEQ ID NO:187, or an amino
acid sequence that is at least at or about 85%, at or about 86%, at
or about 87%, at or about 88%, at or about 89%, at or about 90%, at
or about 91%, at or about 92%, at or about 93%, at or about 94%, at
or about 95%, at or about 96%, at or about 97%, at or about 98% or
at or about 99% sequence identity to the amino acid sequence set
forth in SEQ ID NO:187. In some of any embodiments, the anti-ROR1
CARs has the amino acid sequence set forth in SEQ ID NO:188, or an
amino acid sequence that is at least at or about 85%, at or about
86%, at or about 87%, at or about 88%, at or about 89%, at or about
90%, at or about 91%, at or about 92%, at or about 93%, at or about
94%, at or about 95%, at or about 96%, at or about 97%, at or about
98% or at or about 99% sequence identity to the amino acid sequence
set forth in SEQ ID NO:188. In some of any embodiments, the
anti-ROR1 CARs has the amino acid sequence set forth in SEQ ID
NO:189, or an amino acid sequence that is at least at or about 85%,
at or about 86%, at or about 87%, at or about 88%, at or about 89%,
at or about 90%, at or about 91%, at or about 92%, at or about 93%,
at or about 94%, at or about 95%, at or about 96%, at or about 97%,
at or about 98% or at or about 99% sequence identity to the amino
acid sequence set forth in SEQ ID NO:189.
[0412] In some of any of the provided embodiments, the anti-ROR1
chimeric antigen receptor is encoded by the sequence set forth in
SEQ ID NO: 156, 157, 158, 159, 160 or 161 or a sequence that
exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the sequence set forth in SEQ ID NO: 156, 157, 158,
159, 160 or 161. In some of any embodiments, the anti-ROR1 CARs is
encoded by the nucleotide sequence set forth in SEQ ID NO:156, or a
nucleotide sequence that is at least at or about 85%, at or about
86%, at or about 87%, at or about 88%, at or about 89%, at or about
90%, at or about 91%, at or about 92%, at or about 93%, at or about
94%, at or about 95%, at or about 96%, at or about 97%, at or about
98% or at or about 99% sequence identity to the nucleotide sequence
set forth in SEQ ID NO:156. In some of any embodiments, the
anti-ROR1 CARs is encoded by the nucleotide sequence set forth in
SEQ ID NO:157, or a nucleotide sequence that is at least at or
about 85%, at or about 86%, at or about 87%, at or about 88%, at or
about 89%, at or about 90%, at or about 91%, at or about 92%, at or
about 93%, at or about 94%, at or about 95%, at or about 96%, at or
about 97%, at or about 98% or at or about 99% sequence identity to
the nucleotide sequence set forth in SEQ ID NO:157. In some of any
embodiments, the anti-ROR1 CARs is encoded by the nucleotide
sequence set forth in SEQ ID NO:158, or a nucleotide sequence that
is at least at or about 85%, at or about 86%, at or about 87%, at
or about 88%, at or about 89%, at or about 90%, at or about 91%, at
or about 92%, at or about 93%, at or about 94%, at or about 95%, at
or about 96%, at or about 97%, at or about 98% or at or about 99%
sequence identity to the nucleotide sequence set forth in SEQ ID
NO:158. In some of any embodiments, the anti-ROR1 CARs is encoded
by the nucleotide sequence set forth in SEQ ID NO:159, or a
nucleotide sequence that is at least at or about 85%, at or about
86%, at or about 87%, at or about 88%, at or about 89%, at or about
90%, at or about 91%, at or about 92%, at or about 93%, at or about
94%, at or about 95%, at or about 96%, at or about 97%, at or about
98% or at or about 99% sequence identity to the nucleotide sequence
set forth in SEQ ID NO:159. In some of any embodiments, the
anti-ROR1 CARs is encoded by the nucleotide sequence set forth in
SEQ ID NO:160, or a nucleotide sequence that is at least at or
about 85%, at or about 86%, at or about 87%, at or about 88%, at or
about 89%, at or about 90%, at or about 91%, at or about 92%, at or
about 93%, at or about 94%, at or about 95%, at or about 96%, at or
about 97%, at or about 98% or at or about 99% sequence identity to
the nucleotide sequence set forth in SEQ ID NO:160. In some of any
embodiments, the anti-ROR1 CARs is encoded by the nucleotide
sequence set forth in SEQ ID NO:161, or a nucleotide sequence that
is at least at or about 85%, at or about 86%, at or about 87%, at
or about 88%, at or about 89%, at or about 90%, at or about 91%, at
or about 92%, at or about 93%, at or about 94%, at or about 95%, at
or about 96%, at or about 97%, at or about 98% or at or about 99%
sequence identity to the nucleotide sequence set forth in SEQ ID
NO:161.
[0413] 2. Exemplary Features
[0414] In some of any of the provided embodiments, the provided
recombinant receptors, e.g., CARs, containing an ROR1-binding
antibody or fragment thereof, binds, such as specifically binds to
ROR1, such as ROR1 on the surface of a cancer cell or a tumor cell.
In any of the embodiments, an antibody or antigen binding fragment,
in the provided CARs, specifically binds ROR1, such as a human
ROR1. Exemplary ROR1-binding recombinant receptors, e.g., CARs, can
exhibit any binding affinity, binding specificity and/or other
features of antigen recognition, such as species cross-reactivity,
as described herein, such as in Section I.A.2. In some embodiments,
a CAR containing an extracellular antigen binding domain comprising
the antibodies or fragments thereof exhibit similar or
substantially the same binding properties and features as the
antibody or fragment thereof. In some cases, the CAR containing an
extracellular antigen binding domain comprising the antibodies or
fragment thereof exhibit different binding properties and features
as the antibody or fragment thereof.
[0415] In some embodiments, the provided CARs specifically bind to
a receptor tyrosine kinase-like orphan receptor 1 (ROR1) protein.
In some of any of the embodiments herein, ROR1 refers to human
ROR1. The observation that an antibody or other binding molecule,
e.g., CAR, binds to ROR1 or specifically binds to ROR1 does not
necessarily mean that it binds to ROR1 from every species. For
example, in some embodiments, features of binding to ROR1, such as
the ability to specifically bind thereto and/or to compete for
binding thereto with a reference receptor, e.g., reference CAR,
containing a reference antibody, and/or to bind with a particular
affinity or compete to a particular degree, in some embodiments,
refers to the ability with respect to a human ROR1 protein and the
antibody may not have this feature with respect to a ROR1 of
another species such as mouse. In some embodiments, the CAR binds
to human ROR1 and binds to ROR1 of another species, such as Rhesus
macaque or macaque. In some embodiments, the CAR or an
antigen-binding fragment thereof binds to human ROR1 and does not
bind to ROR1 of another species, such as mouse. In some
embodiments, the CAR binds to human ROR1 and binds to ROR1 of
another species, such as mouse.
[0416] In some embodiments, the CARs bind, such as specifically
bind, to human ROR1, such as to an epitope or region of human ROR1,
such as the human ROR1 set forth in SEQ ID NO:144 (GenBank No.
AAA60275.1; sequence including the signal peptide set forth in SEQ
ID NO:215, Uniprot No. Q01973), or an allelic variant or splice
variant thereof. In one embodiment, human ROR1 is a transcript
variant or isoform that has the sequence of amino acids forth in
SEQ ID NO:145 or 146. In some embodiments, human ROR1 protein
comprises an amino acid sequence set forth in SEQ ID NO: 144, 145,
146 or 215. In some embodiments, the CARs bind to the extracellular
region ROR1, such as to one or more extracellular epitopes present
within the extracellular region of human ROR1, e.g., corresponding
to residues 1-377 of the human ROR1 sequence set forth in SEQ ID
NO:144 (corresponding to residues 30-406 of the human ROR1 sequence
set forth in SEQ ID NO:215 that includes the signal peptide).
[0417] In some embodiments, the CAR binds a linear epitope of ROR1,
such as a human ROR1. In some embodiments, the one or more epitopes
comprises a conformational epitope. In some embodiments, the
antibodies or antigen-binding fragment thereof bind one or more
conformational epitopes of ROR1, such as a human ROR1. In some
embodiments, the antibodies or antigen-binding fragment thereof
bind one or more epitopes of human ROR1, such as one or more
epitopes that include the sequence FRSTIYGSRLRIRNL (set forth in
SEQ ID NO:199) or the sequence set forth in any one of SEQ ID
NO:200-214 or in Table E2 herein. In some embodiments, the
antibodies or antigen-binding fragment thereof bind one or more
epitopes of human ROR1, such as one or more epitopes that include
the sequence FRSTIYGSRLRIRNL (set forth in SEQ ID NO:199). In some
embodiments, the antibodies or antigen-binding fragment thereof
bind additional epitopes, such as one or more conformational
epitopes, in addition to the sequence FRSTIYGSRLRIRNL (set forth in
SEQ ID NO:199). Exemplary of one or more additional epitopes
include, but are not limited to, one or more of the sequence set
forth in any one of SEQ ID NO:200-214 or in Table E2 herein.
[0418] In some embodiments, the antibody binds to non-human ROR1,
such as Rhesus macaques (Macaca mulatta) ROR1 (set forth in SEQ ID
NO:216, Uniprot No. F6RUP2) or cynomolgus macaques (Macaca
fasicularis) ROR1 (set forth in SEQ ID NO:217, Uniprot No.
A0A2K5WTX7; or SEQ ID NO:218, Uniprot No. A0A2K5WTX4). In some
aspects, the extracellular domain of the non-human ROR1 is at least
99% identical to the human ROR1 sequence.
[0419] In some embodiments, the CAR binds to non-human ROR1, such
as monkey, rabbit, rat, mouse, or other species of ROR1. In some
embodiments, the CAR binds to mouse (Mus musculus) ROR1, such as to
an epitope or region of mouse ROR1, such as the mouse ROR1 set
forth in SEQ ID NO: 171 (GenBank No. NP_038873; sequence including
the signal peptide set forth in SEQ ID NO:219, Uniprot No. Q9Z139).
In some embodiments, the CAR binds to human ROR1 and binds to mouse
ROR1. In some embodiments, the extent of binding of some of the
provided anti-ROR1 antibodies or fragments thereof to a non-human
ROR1, such as mouse ROR1, is at least at or about 75%, 80%, 90%,
95%, 100%, 110%, 120%, 130%, 140%, 150% or more of the binding of
the CAR to human ROR1. In some embodiments, the antibodies do not
bind to mouse ROR1, such as the mouse ROR1 set forth in SEQ ID
NO:171.
[0420] In some of any of the provided embodiments, the extent of
binding of the CAR, e.g., containing a ROR1-binding antibody or
antigen-binding fragment thereof does not bind to, is not
cross-reactive to, or binds at a lower extent, level or degree or
affinity to a non-human ROR1, optionally a mouse ROR1. In some
embodiments, the extent of binding of an anti-ROR1 antibody to an
unrelated, non-ROR1 protein or to a non-human ROR1 protein, such as
a mouse ROR1 protein, or other non-ROR1 protein, is less than at or
about 50%, 40%, 30%, 20% or 10% of the binding of the CAR to human
ROR1 as measured. In some embodiments, the antibodies or
antigen-binding fragments thereof do not bind to mouse ROR1, such
as the mouse ROR1 set forth in SEQ ID NO:171 or 219.
[0421] In some of any of the provided embodiments, the CAR or
antigen-binding fragment thereof does not bind to, is not
cross-reactive to, or binds at a lower extent, level or degree or
affinity to a receptor tyrosine kinase-like orphan receptor 2
(ROR2) protein, optionally a human ROR2 protein. In some
embodiments, the extent of binding of some of the provided CAR,
e.g., containing a ROR1-binding antibody or fragment thereof to a
non-ROR1 protein, such as a ROR2 protein, is at least at or about
75%, 80%, 90%, 95% or 99% less than the binding of the CAR to human
ROR1. In some embodiments, the provided antibody or antigen-binding
fragment thereof does not bind to, is not cross-reactive to, or
binds at a lower level or degree or affinity to a ROR2 protein,
optionally a human ROR2 protein. In some embodiments, among
provided CAR, e.g., containing a ROR1-binding antibody or fragment
thereof, are CARs in which binding to mouse ROR1 is less than or at
or about 30%, 20% or 10%, such as less than at or about 10%, of the
binding of the CAR to human ROR1. In some embodiments, among
provided CAR, e.g., containing a ROR1-binding antibody or fragment
thereof, are CARs in which binding to a ROR2, such as a human ROR2,
is less than or at or about 30%, 20% or 10%, such as less than at
or about 10%, of the binding of the CAR to human ROR1. In some
embodiments, the provided CARs exhibit the same, substantially the
same or lower level or degree or affinity of binding to a ROR2
protein compared to a reference ROR1-specific chimeric antigen
receptor, optionally under the same or substantially the same
conditions or assay. In some aspects, binding affinity and/or
specificity to a particular antigen (e.g., human ROR1) can be
assessed using any methods for assessing binding, such as any
described in Section I.A.2 herein.
[0422] In some embodiments, the provided CARs are capable of
binding ROR1, such as human ROR1, with at least a certain affinity,
as measured by any of a number of known methods. In some
embodiments, the affinity is represented by an equilibrium
dissociation constant (K.sub.D). In some embodiments, the affinity
is represented by EC.sub.50.
[0423] In some embodiments, the binding molecule, e.g., CAR, binds,
such as specifically binds, to an antigen, e.g., a ROR1 protein or
an epitope therein, with an affinity or K.sub.A (i.e., an
equilibrium association constant of a particular binding
interaction with units of 1/M or M.sup.-1; equal to the ratio of
the on-rate [k.sub.on or k.sub.a] to the off-rate [k.sub.off or
k.sub.d] for this association reaction, assuming bimolecular
interaction), the binding affinity (EC.sub.50) and/or the
equilibrium dissociation constant (K.sub.D; i.e., an equilibrium
dissociation constant of a particular binding interaction with
units of M; equal to the ratio of the off-rate [k.sub.off or
k.sub.d] to the on-rate [k.sub.on or k.sub.a] for this association
reaction, assuming bimolecular interaction), or an off-rate
(dissociation rate constant; k.sub.off or k.sub.d) as described
herein, e.g., in Section I.A.2 herein. For example, the equilibrium
dissociation constant K.sub.D can range from 10.sup.-5 M to
10.sup.-13 M, such as 10.sup.-7M to 10.sup.-11 M, 10.sup.-7 M to
10.sup.-10 M, 10.sup.-7 M to 10.sup.-9 M, 10.sup.-8 M to 10.sup.-10
M, or 10.sup.-9 M to 10.sup.-10 M.
[0424] In certain embodiments, the binding affinity (EC.sub.50)
and/or the equilibrium dissociation constant, K.sub.D, of the
binding molecule, e.g., anti-ROR1 CAR, to a ROR1 protein, such as a
human ROR1 protein, is at or about or less than at or about 1
.mu.M, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18
nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8
nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less, or a range
defined by any of the foregoing. In some embodiments, the EC.sub.50
and/or the K.sub.D of the binding molecule, e.g., anti-ROR1 CAR, to
a ROR1 protein, is between at or about 10 nM and at or about 90 nM,
between at or about 20 nM and at or about 80 nM, between at or
about 30 nM and at or about 70 nM, between at or about 40 nM and at
or about 60 nM, or between at or about 40 nM and at or about 50 nM.
In certain embodiments, the EC.sub.50 and/or the K.sub.D of the
binding molecule, e.g., anti-ROR1 CAR, to a ROR1 protein, such as a
human ROR1 protein, is at or about 10 nM, 20 nM, 30 nM, 40 nM, 50
nM, 60 nM, 70 nM, 80 nM, 90 nM or 100 nM, or a range defined by any
of the foregoing. In certain embodiments, the EC.sub.50 and/or the
K.sub.D of the binding molecule, e.g., anti-ROR1 CAR, to a ROR1
protein, such as a human ROR1 protein, is at or about 10 nM, 20 nM,
30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM or 100 nM, or a
range defined by any of the foregoing. In certain embodiments, the
EC.sub.50 and/or the K.sub.D of the binding molecule, e.g.,
anti-ROR1 CAR, to a ROR1 protein, such as a human ROR1 protein, is
at or about 40 nM, 41 nM, 42 nM, 43 nM, 44 nM, or 45 nM, or a range
defined by any of the foregoing.
[0425] In some embodiments, the provided binding molecule, e.g.,
anti-ROR1 CAR, has a fast off-rate (dissociation rate constant;
k.sub.off or k.sub.d; units of 1/s or s.sup.-1). In some
embodiments, the off-rate (k.sub.off or k.sub.d) of the provided
binding molecules is between at or about 1.times.10.sup.-5 s.sup.-1
and at or about 1.times.10.sup.-2 s.sup.-1, such as at or about
5.times.10.sup.-5 s.sup.-1 and at or about 9.times.10.sup.-3
s.sup.-1, at or about 1.times.10 s.sup.-1 and at or about
8.times.10.sup.-3 s.sup.-1, at or about 5.times.10.sup.-4 s.sup.-1
and at or about 7.times.10.sup.-3 s.sup.-1, at or about
1.times.10.sup.-3 s.sup.-1 and at or about 6.times.10.sup.-3
s.sup.-1, and at or about 4.times.10.sup.-3 s.sup.-1 and at or
about 6.times.10.sup.-3 s.sup.-1. In some embodiments, the off-rate
(k.sub.off or k.sub.d) of the provided binding molecules is at
least at or about 1.times.10.sup.-5 s.sup.-1, 5.times.10.sup.-5
s.sup.-1, 1.times.10.sup.-4 s.sup.-1, 5.times.10.sup.-4 s.sup.-1,
1.times.10.sup.-3 s.sup.-1, 5.times.10.sup.-3 s.sup.-1, or
1.times.10.sup.-2 s.sup.-1. In some embodiments, the off-rate
(k.sub.off or k.sub.d) of the provided binding molecules is at
least at or about 6.times.10.sup.-4 s.sup.-1, 7.times.10.sup.-4
s.sup.-1, 8.times.10.sup.-4 s.sup.-1, 9.times.10.sup.-4 s.sup.-1,
1.times.10.sup.-3 s.sup.-1, 2.times.10.sup.-3 s.sup.-1,
3.times.10.sup.-3 s.sup.-1, 4.times.10.sup.-3 s.sup.-1,
5.times.10.sup.-3 s.sup.-1, 6.times.10.sup.-3 s.sup.-1,
7.times.10.sup.-3 s.sup.-1, 8.times.10.sup.-3 s.sup.-1,
9.times.10.sup.-3 s.sup.-1 or 1.times.10.sup.-2 s.sup.-1. In some
embodiments, the off-rate (k.sub.off or k.sub.d) of the provided
binding molecules is at least at or about 4.times.10.sup.-3
s.sup.-1, 5.times.10.sup.-3 s or 6.times.10.sup.-3 s.sup.-1, or a
range defined by any of the foregoing. In some embodiments, the
provided binding molecule, e.g., anti-ROR1 CAR, has an off-rate
that is at least at or about 2-fold, 3-fold, 4-fold, 5-fold,
6-fold, 7-fold, 8-fold, 9-fold or 10-fold faster than the off-rate
of a reference anti-ROR1 CAR, for example, anti-ROR1 CAR R12.
[0426] In some embodiments, the binding affinity of a binding
molecule, such as an anti-ROR1 CAR, for different antigens, e.g.,
ROR1 proteins from different species can be compared to determine
the species cross-reactivity. For example, species cross-reactivity
can be classified as high cross reactivity or low cross reactivity.
In some embodiments, the equilibrium dissociation constant,
K.sub.D, for different antigens, e.g., ROR1 proteins from different
species such as human, cynomolgus monkey or mouse, can be compared
to determine species cross-reactivity. In some embodiments, the
species cross-reactivity of an anti-ROR1 CAR can be high, e.g., the
anti-ROR1 CAR binds to human ROR1 and a species variant ROR1 to a
similar degree, e.g., the ratio of K.sub.D for human ROR1 and
K.sub.D for the species variant ROR1 is or is about 1. In some
embodiments, the species cross-reactivity of an anti-ROR1 CAR can
be low, e.g., the anti-ROR1 CAR has a high affinity for human ROR1
but a low affinity for a species variant ROR1, or vice versa. For
example, the ratio of K.sub.D for the species variant ROR1 and
K.sub.D for the human ROR1 is more than 10, 15, 20, 25, 30, 40, 50,
60, 70, 80, 90, 100, 200, 500, 1000, 2000 or more, and the
anti-ROR1 CAR has low species cross-reactivity. The degree of
species cross-reactivity can be compared with the species
cross-reactivity of a known CAR, such as a reference CAR.
[0427] In some embodiments, properties or features of the provided
CARs are described in relation to properties observed for another
CAR, e.g., a reference CAR. In some embodiments, the reference CAR
contains, as an extracellular antigen-binding domain, a reference
antibody described herein or fragment thereof, such as, the
chimeric rabbit/human IgG1 antibody designated R12 (see, e.g., Yang
et al. (2011) PloS ONE, 6:e21018; U.S. Patent Application No. US
2013/0251642); mouse anti-human ROR1 antibody designated 2A2 (see,
e.g., Baskar et al. (2012) MAbs, 4:349-361; published U.S. Patent
Appl. No. US2012/20058051); humanized anti-ROR1 antibodies
described in International PCT Appl. No. WO2014/031174; humanized
variant of an antibody designated 99961.
[0428] In some embodiments, the CAR has an affinity that is about
the same as or lower than that of the corresponding form of the
reference CAR, e.g., EC.sub.50 or K.sub.D that is no more than at
or about 1.5-fold or no more than at or about 2-fold greater, no
more than at or about 3-fold greater, and/or no more than at or
about 10-fold greater, than the EC.sub.50 or K.sub.D of the
corresponding form of the reference CAR. In some embodiments, the
CAR has an affinity that is about the same as or lower than that of
the corresponding form of the reference CAR, e.g., EC.sub.50 or
K.sub.D that is at least at or about 1.5-fold greater, at least at
or about 2-fold greater, at least at or about 3-fold greater, at
least at or about 5-fold greater, at least at or about 10-fold
greater, at least at or about 20-fold greater, at least at or about
25-fold greater, at least at or about 30-fold greater, at least at
or about 40-fold greater, at least at or about 50-fold greater, or
at least at or about 100-fold greater, than the EC.sub.50 or
K.sub.D of the corresponding form of the reference CAR. In some
embodiments, the CAR has an affinity that is about the same as or
lower than that of the corresponding form of the reference CAR, an
affinity that is at or about 1.5-fold, 2-fold, 3-fold, 4-fold,
5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold,
30-fold, 40-fold, 50-fold or 100-fold lower than the affinity of
the reference CAR.
[0429] In some embodiments, the CAR has an affinity that is greater
than that of the corresponding form of the reference CAR, e.g.,
EC.sub.50 or K.sub.D that is lower than or lower than at or about
5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold,
70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold
or lower than the EC.sub.50 or K.sub.D of the corresponding form of
the reference CAR.
[0430] In some embodiments, the CAR specifically binds to an
epitope that overlaps with the epitope of ROR1 bound by a reference
CAR. In some aspects, among such CARs are CARs that bind to the
same or a similar epitope as the reference CAR. In some
embodiments, the CARs bind to the same or a similar epitope or an
epitope within the same region or containing residues within the
same region of ROR1 as a reference CAR, such as anti-ROR1 CAR R12
or scFv fragment thereof (set forth in SEQ ID NO: 142; see e.g.
Yang et al. (2011) PloS ONE, 6:e21018). In some embodiments, the
CAR inhibits binding to and/or competes for binding to ROR1, such
as human ROR1, with the reference CAR.
[0431] Among the provided CARs are CARs that exhibit
antigen-dependent activity or signaling, i.e. signaling activity
that is measurably absent or at background levels in the absence of
antigen, e.g. ROR1, and/or in the presence of non-specific antigen.
Thus, in some aspects, provided CARs do not exhibit, or exhibit no
more than background or a tolerable or low level of, tonic
signaling or antigen-independent activity or signaling in the
absence of antigen, e.g. ROR1, being present. In some embodiments,
the provided anti-ROR1 CAR-expressing cells exhibit biological
activity or function, including cytotoxic activity, cytokine
production, and ability to proliferate. In some embodiments, the
provided CARs receptor exhibits the same, substantially the same or
higher antigen-specific signaling and/or antigen dependent activity
or signaling compared to a reference ROR1-specific chimeric antigen
receptor, optionally under the same or substantially the same
conditions or assay.
[0432] In some embodiments, the provided CARs exhibit absent,
reduced or lower levels of activity or signaling in the absence of
antigen, e.g., ROR1. In some embodiments, the provided CARs exhibit
reduced, lower or is nearly absent of or completely absent of tonic
signaling or antigen-independent signaling or activity, e.g.,
signaling or activity in the absence of antigen, such that the
signaling or activity is less than or at or about 30%, 20% or 10%,
such as less than at or about 10%, of the signaling or activity of
the CAR in the presence of human ROR1. In some embodiments, the
provided CARs exhibit the same, substantially the same or lower
tonic signaling and/or antigen independent activity or signaling
compared to a reference ROR1-specific chimeric antigen receptor
(CAR), optionally under the same or substantially the same
conditions or assay. In some aspects, among a plurality or
population of cells that are engineered to express the CARs, less
than at or about 10%, at or about 9%, at or about 8%, at or about
7%, at or about 5%, at or about 4%, at or about 3%, at or about 2%
or at or about 1% of the cells in the plurality comprise a chimeric
antigen receptor that exhibits tonic signaling and/or antigen
independent activity or signaling. In some embodiments, reference
ROR1-specific CARs include those that have an antigen-binding
domain that comprises any reference anti-ROR1 antibodies described
herein, such as those described in Section I.A.2 herein, for
example, R12, A2A or 99961, or an antigen-binding fragment
thereof.
[0433] In some embodiments, the provided CARs exhibit absent,
reduced or lower levels of activity or signaling when exposed to a
non-specific antigen, such as a different but related protein, such
as ROR2. In some embodiments, the provided CARs exhibit the same,
substantially the same or lower level or degree or affinity of
binding to a ROR2 protein compared to a reference ROR1-specific
chimeric antigen receptor, optionally under the same or
substantially the same conditions or assay. In some embodiments,
the provided CARs exhibit reduced signaling or activity in the
presence of a ROR2, such as a human ROR2, such that the signaling
or activity is less than or at or about 30%, 20% or 10%, such as
less than at or about 10%, of the signaling or activity of the CAR
in the presence of human ROR1.
[0434] In some embodiments, engineered cells expressing the
provided anti-ROR1 CARs exhibit improved biological activity or
functional activity, such as anti-tumor activity, tumor growth
inhibition, tumor volume reduction, persistence, expansion, or
prolonged survival of the subject, when administered to a subject
for adoptive cell therapy. In some embodiments, engineered cells
expressing the provided CARs exhibit improved biological activity
or functional activity, such as anti-tumor activity, tumor growth
inhibition, tumor volume reduction, persistence, expansion, or
prolonged survival of the subject, compared to engineered cells
expressing a reference ROR1-specific chimeric antigen receptor,
optionally under the same or substantially the same conditions or
assay. In some embodiments, biological activity or functional
activity of a chimeric receptor, such as cytotoxic activity, can be
measured using any of a number of known methods. The activity can
be assessed or determined either in vitro or in vivo. In some
embodiments, activity can be assessed once the cells are
administered to the subject (e.g., human) Parameters to assess
include specific binding of an engineered or natural T cell or
other immune cell to antigen, e.g., in vivo, e.g., by imaging, or
ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments,
the ability of the engineered cells to destroy target cells can be
measured using any suitable known methods, such as cytotoxicity
assays described in, for example, Kochenderfer et al., J.
Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J.
Immunological Methods, 285(1): 25-40 (2004). In certain
embodiments, the biological activity of the cells also can be
measured by assaying expression and/or secretion of certain
cytokines, such as interlekukin-2 (IL-2), interferon-gamma
(IFN.gamma.), interleukin-4 (IL-4), TNF-alpha (TNF.alpha.),
interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12
(IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF),
CD107a, and/or TGF-beta (TGF.beta.). Assays to measure cytokines
are well known, and include but are not limited to, ELISA,
intracellular cytokine staining, cytometric bead array, RT-PCR,
ELISPOT, flow cytometry and bio-assays in which cells responsive to
the relevant cytokine are tested for responsiveness (e.g.
proliferation) in the presence of a test sample. In some aspects
the biological activity can be measured using an animal model of
the disease or condition, such as a tumor xenograft model, and
assessing the reduction in tumor burden or load and/or survival. In
some aspects the biological activity is measured by assessing
clinical outcome, such as reduction in tumor burden or load.
[0435] In some embodiments, administration of engineered cells
expressing the provided anti-ROR1 CARs exhibit substantially
improved tumor growth inhibition, in vivo persistence and/or
prolonged survival of the subject with a tumor, compared to
administration of engineered cells expressing reference CAR, such
as anti-ROR1 R12. In some aspects, such improvement is observed in
subjects having various types of ROR1-expressing cancers,
including, but not limited to, lung cancer, breast cancer, chronic
lymphocytic leukemia (CLL), ovarian cancer or mantle cell lymphoma
(MCL).
[0436] In some embodiments, engineered cells expressing the
provided anti-ROR1 CARs exhibit improved persistence and expansion
when administered to a subject for adoptive cell therapy. In some
embodiments, engineered cells expressing the provided CARs exhibit
the same, substantially the same or higher persistence and
expansion compared to engineered cells expressing a reference
ROR1-specific chimeric antigen receptor, optionally under the same
or substantially the same conditions or assay. In some aspects, the
proliferation, expansion and/or persistence of an engineered cell
expressing any of the provided receptors, e.g., CARs, can be
assessed by determining the exposure, number, concentration,
persistence and proliferation of the engineered cells, e.g., cells
administered for adoptive cell therapy. In some embodiments, the
exposure, number or level of engineered T cells, e.g., T cells
administered for the T cell based therapy, or subset thereof, such
as CD3.sup.+ cells, CD4.sup.+ cells, CD8.sup.+ cells, CD3.sup.+
CAR.sup.+ cells, CD4.sup.+ CAR.sup.+ cells or CD8.sup.+ CAR.sup.+
cells can be assessed, e.g., from a subject, such as a human
subject or an animal subject, that had been administered engineered
cells. In some aspects, the exposure, number, concentration,
persistence and proliferation relate to pharmacokinetic parameters.
In some cases, pharmacokinetics can be assessed by measuring such
parameters as the maximum (peak) plasma concentration (C.sub.max),
the peak time (i.e. when maximum plasma concentration (C.sub.max)
occurs; T.sub.max), the minimum plasma concentration (i.e. the
minimum plasma concentration between doses of a therapeutic agent,
e.g., CAR.sup.+ T cells; C.sub.min), the elimination half-life
(T.sub.1/2) and area under the curve (i.e. the area under the curve
generated by plotting time versus plasma concentration of the
therapeutic agent CAR.sup.+ T cells; AUC), following
administration. The concentration of a particular therapeutic
agent, e.g., CAR.sup.+ T cells, in the plasma following
administration can be measured using any known methods suitable for
assessing concentrations of the therapeutic agents, e.g., CAR.sup.+
T cells, in samples of blood, or any methods described herein. For
example, nucleic acid-based methods, such as quantitative PCR
(qPCR) or flow cytometry-based methods, or other assays, such as an
immunoassay, ELISA, or chromatography/mass spectrometry-based
assays can be used.
[0437] In some aspects, a reporter cell line can be employed to
monitor antigen-independent activity and/or tonic signaling through
anti-ROR1 CAR-expressing cells. In some embodiments, a T cell line,
such as a Jurkat cell line, contains a reporter molecule, such as a
fluorescent protein or other detectable molecule, such as a red
fluorescent protein, expressed under the control of the endogenous
Nur77 transcriptional regulatory elements. In some embodiments, the
Nur77 reporter expression is cell intrinsic and dependent upon
signaling through a recombinant reporter containing a primary
activation signal in a T cell, a signaling domain of a T cell
receptor (TCR) component, and/or a signaling domain comprising an
immunoreceptor tyrosine-based activation motif (ITAM), such as a
CD3 chain. Nur77 expression is generally not affected by other
signaling pathways such as cytokine signaling or toll-like receptor
(TLR) signaling, which may act in a cell extrinsic manner and may
not depend on signaling through the recombinant receptor. Thus,
only cells that express the exogenous recombinant receptor, e.g.,
anti-ROR1 CAR, containing the appropriate signaling regions is
capable of expressing Nur77 upon stimulation (e.g., binding of the
specific antigen). In some cases, Nur77 expression also can show a
dose-dependent response to the amount of stimulation (e.g.,
antigen).
[0438] In some cases, to assess the specificity, cross-reactivity
and/or antigen dependency of a particular activity, signaling or
function of the receptor, e.g., CAR, any of the described assays
for assessing activity, signaling or biological function of the
receptors can be assessed in the presence and absence of the
specific target antigen, e.g., human ROR1, or in the presence of
the specific target antigen, e.g., human ROR1, and in the presence
of a different, non-specific antigen, e.g., human ROR2 or a
non-human ROR1, such as a mouse ROR1.
[0439] In some of any embodiments, the provided anti-ROR1 CAR
exhibits the same, substantially the same or lower level or degree
of signaling or activity in the presence of a ROR2 protein, e.g., a
human ROR2, compared to the level or degree of signaling or
activity in the presence of a ROR1 protein, e.g., a human ROR1,
e.g., under the same or substantially the same conditions or assay.
For example, in some aspects, the provided anti-ROR1 CAR exhibits a
level or degree of signaling or activity in the presence of a human
ROR2 that is at least at or about 75%, 80%, 90%, 95% or 99% less
than the level or degree of signaling or activity in the presence
of a human ROR1, e.g., under the same or substantially the same
conditions or assay.
[0440] In some of any embodiments, the provided anti-ROR1 CAR
exhibits the same, substantially the same or lower level or degree
of signaling or activity in the presence of a ROR2 protein, e.g., a
human ROR2, compared to a reference ROR1-specific CAR, e.g., under
the same or substantially the same conditions or assay. In some of
any embodiments, the provided anti-ROR1 CAR exhibits the same,
substantially the same or lower level or degree of signaling or
activity in the presence of a ROR2 protein, e.g., a human ROR2,
compared to a reference ROR1-specific CAR, e.g., under the same or
substantially the same conditions or assay. In some of any
embodiments, the provided anti-ROR1 CAR exhibits the same,
substantially the same or higher antigen-specific signaling and/or
antigen dependent activity or signaling compared to a reference
ROR1-specific CAR, e.g., under the same or substantially the same
conditions or assay.
[0441] In some of any embodiments, the provided anti-ROR1 CAR
exhibits the same, substantially the same or lower tonic signaling
and/or antigen independent activity or signaling compared to a
reference ROR1-specific CAR, e.g., under the same or substantially
the same conditions or assay. For example, in some embodiments, the
provided anti-ROR1 CAR exhibits a level or degree of tonic
signaling and/or antigen independent activity or signaling that is
at least at or about 75%, 80%, 90%, 95% or 99% less than the level
or degree of tonic signaling and/or antigen independent activity of
a reference ROR1-specific CAR, e.g., under the same or
substantially the same conditions or assay.
[0442] In some embodiments, the provided anti-ROR1 CARs exhibit
improved expression on the surface of cells, such as compared to an
alternative CAR that has an identical amino acid sequence but that
is encoded by non-splice site eliminated and/or a codon-optimized
nucleotide sequence. In some embodiments, the expression of the
recombinant receptor on the surface of the cell can be assessed.
Approaches for determining expression of the recombinant receptor
on the surface of the cell may include use of chimeric antigen
receptor (CAR)-specific antibodies (e.g., Brentjens et al., Sci.
Transl. Med. 2013 March; 5(177): 177ra38), Protein L (Zheng et al.,
J. Transl. Med. 2012 February; 10:29), epitope tags, and monoclonal
antibodies that specifically bind to a CAR polypeptide (see
WO2014190273). In some embodiments, the expression of the
recombinant receptor on the surface of the cell, e.g., primary T
cell, can be assessed, for example, by flow cytometry, using
binding molecules that can bind to the recombinant receptor or a
portion thereof that can be detected. In some embodiments, the
binding molecules used for detecting expression of the recombinant
receptor an anti-idiotypic antibody, e.g., an anti-idiotypic
agonist antibody specific for a binding domain, e.g., scFv, or a
portion thereof. In some embodiments, the binding molecule is or
comprises an isolated or purified antigen, e.g., recombinantly
expressed antigen.
[0443] E. Polynucleotides Encoding Binding Molecules
[0444] Also provided are polynucleotides encoding the binding
molecules, such as anti-ROR1 antibodies, antigen-binding fragments
thereof, recombinant receptors (e.g., chimeric antigen receptors)
and/or portions, e.g., chains or fragments, thereof. Among the
provided polynucleotides are those encoding the anti-ROR1
antibodies (e.g., antigen-binding fragment) or chimeric antigen
receptors described herein. The polynucleotides may include those
encompassing natural and/or non-naturally occurring nucleotides and
bases, e.g., including those with backbone modifications. The terms
"nucleic acid molecule", "nucleic acid", "sequence of nucleotides",
and "polynucleotide" may be used interchangeably, and refer to a
polymer of nucleotides. Such polymers of nucleotides may contain
natural and/or non-natural nucleotides, and include, but are not
limited to, DNA, RNA, and PNA. "Nucleic acid sequence" refers to
the linear sequence of nucleotides that comprise the nucleic acid
molecule or polynucleotide.
[0445] Provided are polynucleotides that contain a nucleic acid
encoding any of the anti-ROR1 antibody or antigen-binding domain
thereof described herein, or any portion, fragment, chain or domain
thereof. In some embodiments, the antibody or antigen-binding
fragment thereof contain multiple domains or chains (e.g., heavy
chain and a light chain), and all of the antibody or
antigen-binding fragment thereof is encoded in one polynucleotide.
Also provided are polynucleotides that contain a nucleic acid
encoding any of the single chain cell surface proteins described
herein. Also provided are polynucleotides that contain a nucleic
acid encoding any of the conjugate described herein. Also provided
are polynucleotides that contain a nucleic acid encoding any of the
anti-ROR1 chimeric antigen receptors described herein. In some
embodiments, the binding molecule, such as the antibody or
antigen-binding fragment thereof or recombinant receptors, contain
multiple domains or chains (e.g., a heavy chain and a light chain),
and all of the binding molecule is encoded in more than one
polynucleotide, such as two or more polynucleotides. In some
embodiments, the polynucleotides are comprised in a vector.
[0446] In some aspects, provided are polynucleotides that contain
nucleic acid sequences encoding any of the binding molecules
provided herein, for example, in Section I.A and I.D. In some
embodiments, provided are polynucleotides that contain nucleic acid
sequences encoding a portion, fragment, chain or domain of any of
the binding molecules provided herein, for example, in Section I.A
and I.D.
[0447] In some cases, the polynucleotide encoding the ROR1-binding
molecules, such as an antibody or antigen-binding fragment thereof
or a recombinant receptor (e.g., CAR) contains a signal sequence
that encodes a signal peptide, in some cases encoded upstream of
the nucleic acid sequences encoding the ROR1-binding molecules,
such as an antibody or antigen-binding fragment thereof or a
recombinant receptor (e.g., CAR), or joined at the 5' terminus of
the nucleic acid sequences encoding the antigen-binding domain. In
some cases, the polynucleotide containing nucleic acid sequences
encoding the ROR1-binding molecules, such as an antibody or
antigen-binding fragment thereof or a recombinant receptor (e.g.,
CAR), contains a signal sequence that encodes a signal peptide. In
some aspects, the signal sequence may encode a signal peptide
derived from a native polypeptide. In other aspects, the signal
sequence may encode a heterologous or non-native signal peptide. In
some aspects, non-limiting exemplary signal peptide include a
signal peptide of the IgG kappa chain set forth in SEQ ID NO: 43 or
encoded by the nucleotide sequence set forth in SEQ ID NO:44. In
some aspects, a non-limiting exemplary signal peptide includes a
signal peptide of a GMCSFR alpha chain set forth in SEQ ID NO:45
and encoded by the nucleotide sequence set forth in SEQ ID NO:46.
In some aspects, a non-limiting exemplary signal peptide includes a
signal peptide of a CD8 alpha signal peptide set forth in SEQ ID
NO:47. In some aspects, a non-limiting exemplary signal peptide
includes a signal peptide of a CD33 signal peptide set forth in SEQ
ID NO:42 and encoded by the nucleotide sequence set forth in SEQ ID
NO:190. In some cases, the polynucleotide encoding the ROR1-binding
molecules, such as an antibody or antigen-binding fragment thereof
or a recombinant receptor (e.g., CAR) can contain nucleic acid
sequence encoding additional molecules, such as a surrogate marker
or other markers, or can contain additional components, such as
promoters, regulatory elements and/or multicistronic elements. In
some embodiments, the nucleic acid sequence encoding the
ROR1-binding molecules, such as an antibody or antigen-binding
fragment thereof or a recombinant receptor (e.g., CAR) can be
operably linked to any of the additional components.
[0448] In some embodiments, provided are polynucleotides contain
nucleic acid sequences encoding a variable heavy chain domain
(V.sub.H) of an antibody or an antigen-binding fragment thereof, or
a recombinant receptor containing an antibody or an antigen-binding
fragment thereof. In some embodiments, provided are polynucleotides
contain nucleic acid sequences encoding a variable light chain
domain (V.sub.L) of an antibody or an antigen-binding fragment
thereof, or a recombinant receptor containing an antibody or an
antigen-binding fragment thereof. In some embodiments, provided are
polynucleotides that contain nucleic acid sequences encoding a
variable heavy chain domain (V.sub.H) and a variable light chain
domain (V.sub.L) of an antibody or an antigen-binding fragment
thereof, or a recombinant receptor containing an antibody or an
antigen-binding fragment thereof.
[0449] In some of any embodiments, said polynucleotide contains a
nucleic acid encoding the V.sub.H comprising the sequence set forth
in SEQ ID NO: 110, 119, 101 or 128 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 110, 119, 101 or 128. In some of any
embodiments, said polynucleotide contains a nucleic acid encoding
the V.sub.L comprising the sequence set forth in SEQ ID NO: 113,
122 or 104, or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 113, 122
or 104.
[0450] In some of any embodiments, said polynucleotide contains a
nucleic acid encoding the V.sub.H comprising the sequence set forth
in SEQ ID NO: 110, 119, 101 or 128 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 110, 119, 101 or 128, and a nucleic acid
encoding the V.sub.L comprising the sequence set forth in SEQ ID
NO: 113, 122 or 104, or a nucleic acid sequence having at least at
or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 113, 122 or 104.
[0451] In some of any embodiments, said polynucleotide contains a
nucleic acid encoding the V.sub.H comprising the sequence set forth
in SEQ ID NO: 110 or a nucleic acid sequence having at least at or
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:
110. In some embodiments, said polynucleotide contains a nucleic
acid encoding the V.sub.L comprising the sequence set forth in SEQ
ID NO: 113, or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 113. In
some of any embodiments, said polynucleotide contains a nucleic
acid encoding the V.sub.H comprising the sequence set forth in SEQ
ID NO: 119 or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 119. In
some embodiments, said polynucleotide contains a nucleic acid
encoding the V.sub.L comprising the sequence set forth in SEQ ID
NO: 122, or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 122.
[0452] In some of any embodiments, said polynucleotide contains a
nucleic acid encoding the V.sub.H comprising the sequence set forth
in SEQ ID NO: 101 or a nucleic acid sequence having at least at or
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:
101. In some embodiments, said polynucleotide contains a nucleic
acid encoding the V.sub.L comprising the sequence set forth in SEQ
ID NO: 104, or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 104. In
some of any embodiments, said polynucleotide contains a nucleic
acid encoding the V.sub.H comprising the sequence set forth in SEQ
ID NO: 128 or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 128. In
some embodiments, said polynucleotide contains a nucleic acid
encoding the V.sub.L comprising the sequence set forth in SEQ ID
NO: 104, or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 104.
[0453] In some of any embodiments, said polynucleotide contains a
nucleic acid encoding the V.sub.H comprising the sequence set forth
in SEQ ID NO: 110 or a nucleic acid sequence having at least at or
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:
110, and a nucleic acid encoding the V.sub.L comprising the
sequence set forth in SEQ ID NO: 113, or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 113. In some of any embodiments, said
polynucleotide contains a nucleic acid encoding the V.sub.H
comprising the sequence set forth in SEQ ID NO: 119 or a nucleic
acid sequence having at least at or about 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 119, and a nucleic acid encoding
the V.sub.L comprising the sequence set forth in SEQ ID NO: 122, or
a nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 122.
[0454] In some of any embodiments, said polynucleotide contains a
nucleic acid encoding the V.sub.H comprising the sequence set forth
in SEQ ID NO: 101 or a nucleic acid sequence having at least at or
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:
101, and a nucleic acid encoding the V.sub.L comprising the
sequence set forth in SEQ ID NO: 104, or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 104. In some of any embodiments, said
polynucleotide contains a nucleic acid encoding the V.sub.H
comprising the sequence set forth in SEQ ID NO: 128 or a nucleic
acid sequence having at least at or about 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 128, and a nucleic acid encoding
the V.sub.L comprising the sequence set forth in SEQ ID NO: 104, or
a nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 104.
[0455] In some of any embodiments, said polynucleotide contains a
nucleic acid encoding the scFv comprising the sequence set forth in
SEQ ID NO: 116, 125, 107 or 132 or a nucleic acid sequence having
at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID NO: 116, 125, 107 or 132. In some of any embodiments, said
polynucleotide contains a nucleic acid encoding the scFv comprising
the sequence set forth in SEQ ID NO: 116 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 116. In some of any embodiments, said
polynucleotide contains a nucleic acid encoding the scFv comprising
the sequence set forth in SEQ ID NO: 125 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 125. In some of any embodiments, said
polynucleotide contains a nucleic acid encoding the scFv comprising
the sequence set forth in SEQ ID NO: 107 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 107. In some of any embodiments, said
polynucleotide contains a nucleic acid encoding the scFv comprising
the sequence set forth in SEQ ID NO: 132 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 132.
[0456] In some of any embodiments, said polynucleotide contains a
nucleic acid encoding the spacer comprising the sequence set forth
in SEQ ID NO: 30 or a nucleic acid sequence having at least at or
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 30.
In some of any embodiments, said polynucleotide contains a nucleic
acid encoding the spacer comprising the sequence set forth in SEQ
ID NO: 192 or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 192. In
some of any embodiments, said polynucleotide contains a nucleic
acid encoding the spacer comprising the sequence set forth in SEQ
ID NO: 193 or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 193. In
some of any embodiments, said polynucleotide contains a nucleic
acid encoding the spacer comprising the sequence set forth in SEQ
ID NO: 195 or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 195.
[0457] Also provided are polynucleotides that have been optimized
for codon usage and/or to eliminate splice sites, such as cryptic
splice sites. In some embodiments, the polynucleotides are modified
to optimize codon usage. In some embodiments, the polynucleotides
are codon optimized for expression in a human cell such as a human
T cell such as a primary human T cell. In some embodiments, the
polynucleotides, such as those encoding any of the antibodies,
receptors (such as antigen receptors such as chimeric antigen
receptors) and/or ROR1-specific binding proteins provided herein,
are or have been modified to reduce heterogeneity or contain one or
more nucleic acid sequences observed herein (such as by the
optimization methods) to result in improved features of the
polypeptides, such as the CARs, as compared to those containing
distinct, reference, sequences or that have not been optimized. In
some embodiments, the polynucleotide is optimized by splice site
elimination. Among such features include improvements in RNA
heterogeneity, such as that resulting from the presence of one or
more splice sites, such as one or more cryptic splice sites, and/or
improved expression and/or surface expression of the encoded
protein, such as increased levels, uniformity, or consistency of
expression among cells or different therapeutic cell compositions
engineered to express the polypeptides. In some embodiments, the
polynucleotides can be codon optimized for expression in human
cells.
[0458] Genomic nucleic acid sequences generally, in nature, in a
mammalian cell, undergo processing co-transcriptionally or
immediately following transcription, wherein a nascent precursor
messenger ribonucleic acid (pre-mRNA), transcribed from a genomic
deoxyribonucleic acid (DNA) sequence, is in some cases edited by
way of splicing, to remove introns, followed by ligation of the
exons in eukaryotic cells. Consensus sequences for splice sites are
known, but in some aspects, specific nucleotide information
defining a splice site may be complex and may not be readily
apparent based on available methods. Cryptic splice sites are
splice sites that are not predicted based on the standard consensus
sequences and are variably activated. Hence, variable splicing of
pre-mRNA at cryptic splice sites leads to heterogeneity in the
transcribed mRNA products upon expression in eukaryotic cells.
[0459] Polynucleotides generated for the expression of transgenes
are typically constructed from nucleic acid sequences, such as
complementary DNA (cDNA), or portions thereof, that do not contain
introns. Thus, splicing of such sequences is not expected to occur.
However, the presence of cryptic splice sites within the cDNA
sequence can lead to unintended or undesired splicing reactions and
heterogeneity in the transcribed mRNA. Such heterogeneity results
in translation of unintended protein products, such as truncated
protein products with variable amino acid sequences that exhibit
modified expression and/or activity.
[0460] In some embodiments, eliminating splice sites, such as
cryptic splice sites, can improve or optimize expression of a
transgene product, such as a polypeptide translated from the
transgene, such as an anti-ROR1 CAR polypeptide. Splicing at
cryptic splice sites of an encoded transgene, such as an encoded
ROR1 CAR molecule, can lead to reduced protein expression, e.g.,
expression on cell surfaces, and/or reduced function, e.g., reduced
intracellular signaling. Provided herein are polynucleotides,
encoding anti-ROR1 CAR proteins that have been optimized to reduce
or eliminate cryptic splice sites. Also provided herein are
polynucleotides encoding anti-ROR1 CAR proteins that have been
optimized for codon expression and/or in which one or more
sequence, such as one identified by the methods or observations
herein regarding splice sites, is present, and/or in which an
identified splice site, such as any of the identified splice sites
herein, is not present. Among the provided polynucleotides are
those exhibiting below a certain degree of RNA heterogeneity or
splice forms when expressed under certain conditions and/or
introduced into a specified cell type, such as a human T cell, such
as a primary human T cell, and cells and compositions and articles
of manufacture containing such polypeptides and/or exhibiting such
properties. In some embodiments, the RNA heterogeneity of
transcribed RNA is reduced by greater than or greater than about
10%, 15%, 20%, 25%, 30%, 40%, 50% or more compared to a
polynucleotide that has not been modified to remove cryptic splice
sites and/or by codon optimization. In some embodiments, the
provided polynucleotides encoding an anti-ROR1 CAR exhibit RNA
homogeneity of transcribed RNA that is at least 70%, 75%, 80%, 85%,
90%, or 95% or greater.
[0461] RNA heterogeneity can be determined by any of a number of
methods provided herein or described or known. In some embodiments,
RNA heterogeneity of a transcribed nucleic acid is determined by
amplifying the transcribed nucleic acid, such as by reverse
transcriptase polymerase chain reaction (RT-PCR) followed by
detecting one or more differences, such as differences in size, in
the one or more amplified products. In some embodiments, the RNA
heterogeneity is determined based on the number of differently
sized amplified products, or the proportion of various differently
sized amplified products. In some embodiments, RNA, such as total
RNA or cytoplasmic polyadenylated RNA, is harvested from cells,
expressing the transgene to be optimized, and amplified by reverse
transcriptase polymerase chain reaction (RT-PCR) using a primer
specific to the 5' untranslated region (5' UTR), in some cases
corresponding to a portion of the promoter sequence in the
expression vector, located upstream of the transgene in the
transcribed RNA, and a primer specific to the 3' untranslated
region (3' UTR), located downstream of the expressed transgene in
the transcribed RNA sequence or a primer specific to a sequence
within the transgene. In particular embodiments, at least one
primer complementary to a sequence in the 5' untranslated region
(UTR) and at least one primer complementary to a sequence in the 3'
untranslated region (UTR) are employed to amplify the transgene.
One can resolve RNA, such as messenger RNA, and analyze the
heterogeneity thereof by several methods. Non-limiting, exemplary
methods include agarose gel electrophoresis, chip-based capillary
electrophoresis, analytical centrifugation, field flow
fractionation, and chromatography, such as size exclusion
chromatography or liquid chromatography.
[0462] In some embodiments, a provided polynucleotide encoding an
anti-ROR1 CAR provided herein, or a construct provided herein,
includes modifications to remove one or more splice donor and/or
acceptor site that may contribute to splice events and/or reduced
expression and/or increased RNA heterogeneity. In some embodiments,
provided polynucleotides are modified in one or more
polynucleotides in the spacer region to eliminate or reduce splice
events.
[0463] In some of any embodiments, said polynucleotide contains a
nucleic acid encoding the V.sub.H comprising the sequence set forth
in SEQ ID NO: 111, 120, 102 or 129, and a nucleic acid encoding the
V.sub.L comprising the sequence set forth in SEQ ID NO: 114, 123,
105 or 131. In some of any embodiments, said polynucleotide
contains a nucleic acid encoding the V.sub.H comprising the
sequence set forth in SEQ ID NO: 111, and a nucleic acid encoding
the V.sub.L comprising the sequence set forth in SEQ ID NO: 114. In
some of any embodiments, said polynucleotide contains a nucleic
acid encoding the V.sub.H comprising the sequence set forth in SEQ
ID NO: 120, and a nucleic acid encoding the V.sub.L comprising the
sequence set forth in SEQ ID NO: 123. In some of any embodiments,
said polynucleotide contains a nucleic acid encoding the V.sub.H
comprising the sequence set forth in SEQ ID NO: 102, and a nucleic
acid encoding the V.sub.L comprising the sequence set forth in SEQ
ID NO: 105. In some of any embodiments, said polynucleotide
contains a nucleic acid encoding the V.sub.H comprising the
sequence set forth in SEQ ID NO: 129, and a nucleic acid encoding
the V.sub.L comprising the sequence set forth in SEQ ID NO:
131.
[0464] In some of any embodiments, said polynucleotide contains a
nucleic acid encoding the scFv comprising the sequence set forth in
SEQ ID NO: 117, 126, 108 or 133. In some of any embodiments, said
polynucleotide contains a nucleic acid encoding the scFv comprising
the sequence set forth in SEQ ID NO: 117. In some of any
embodiments, said polynucleotide contains a nucleic acid encoding
the scFv comprising the sequence set forth in SEQ ID NO: 126. In
some of any embodiments, said polynucleotide contains a nucleic
acid encoding the scFv comprising the sequence set forth in SEQ ID
NO: 108. In some of any embodiments, said polynucleotide contains a
nucleic acid encoding the scFv comprising the sequence set forth in
SEQ ID NO: 133.
[0465] In some of any embodiments, said polynucleotide contains a
nucleic acid encoding the spacer comprising the sequence set forth
in SEQ ID NO:136. In some of any embodiments, said polynucleotide
contains a nucleic acid encoding the spacer comprising the sequence
set forth in SEQ ID NO:139. In some of any embodiments, said
polynucleotide contains a nucleic acid encoding the spacer
comprising the sequence set forth in SEQ ID NO:196.
[0466] Provided in are exemplary chimeric antigen receptors (CARs)
specific for ROR1, and polynucleotides containing nucleic acid
sequences encoding all or a portion, fragment, domain or chain of
any of the exemplary CARs described herein. In some embodiments,
the exemplary CARs contain one of the ROR1-binding antibody
fragment, such as those described in Section I.A and/or Table 2. In
some embodiments, the exemplary CARs are among those described in
each row of Table E1 and/or Table 3. Also provided herein are
polynucleotides encoding the CARs. In some embodiments, the CAR can
be encoded by more than one different polynucleotides, such as two
or more polynucleotides. In some of any such embodiments, two or
more polynucleotides can each contain nucleic acids encoding a
portion, fragment, domain or chain of the CAR.
[0467] Also provided herein are exemplary modified polynucleotides,
including polynucleotides that were modified for codon optimization
(0) and/or splice site elimination (SSE). Examples of the SEQ ID
NOS: for such polynucleotides are set forth in Table 3, wherein
exemplary nucleotide (nt) sequences for the components of the
exemplary CAR constructs prior to splice site elimination and codon
optimization (N/O), nucleic acid (nt) sequences for the components
of the CAR constructs following splice site elimination and
optimization (O/SSE), and the corresponding amino acid (aa)
sequences encoded by the nucleic acid sequences are provided. The
components include the anti-ROR1 scFv, spacer, transmembrane (tm)
domain, 4-1BB costimulatory signaling region sequence (costim),
CD3.zeta. signaling domain (CD3.zeta.). The full sequences of the
exemplary CARs following splice site elimination and optimization
(CAR) are also provided. In some cases, the polynucleotide also
includes the CD33 signal sequence (ss), a T2A ribosomal skip
element (T2A) and truncated EGF receptor (EGFRt) sequence.
Polynucleotide sequences of exemplary CAR are set forth in SEQ ID
NOs: 156-161, encoding the amino acid sequences set forth in SEQ ID
NOs: 184-189.
[0468] In some of any embodiments, said polynucleotide contains the
sequence set forth in SEQ ID NO: 156, 157, 158, 159, 160 or 161 or
a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% sequence identity to the sequence set forth in SEQ ID NO: 156,
157, 158, 159, 160 or 161. In some of any embodiments, said
polynucleotide contains the sequence set forth in SEQ ID NO: 156 or
a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% sequence identity to the sequence set forth in SEQ ID NO: 156.
In some of any embodiments, said polynucleotide contains the
sequence set forth in SEQ ID NO: 156. In some of any embodiments,
said polynucleotide contains the sequence set forth in SEQ ID NO:
157 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the sequence set forth in SEQ ID
NO: 157. In some of any embodiments, said polynucleotide contains
the sequence set forth in SEQ ID NO: 157. In some of any
embodiments, said polynucleotide contains the sequence set forth in
SEQ ID NO: 158 or a sequence that exhibits at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% sequence identity to the sequence set forth
in SEQ ID NO: 158. In some of any embodiments, said polynucleotide
contains the sequence set forth in SEQ ID NO: 158. In some of any
embodiments, said polynucleotide contains the sequence set forth in
SEQ ID NO: 159 or a sequence that exhibits at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% sequence identity to the sequence set forth
in SEQ ID NO: 159. In some of any embodiments, said polynucleotide
contains the sequence set forth in SEQ ID NO: 159. In some of any
embodiments, said polynucleotide contains the sequence set forth in
SEQ ID NO: 160 or a sequence that exhibits at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% sequence identity to the sequence set forth
in SEQ ID NO: 160. In some of any embodiments, said polynucleotide
contains the sequence set forth in SEQ ID NO: 160. In some of any
embodiments, said polynucleotide contains the sequence set forth in
SEQ ID NO: 161 or a sequence that exhibits at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% sequence identity to the sequence set forth
in SEQ ID NO: 161. In some of any embodiments, said polynucleotide
contains the sequence set forth in SEQ ID NO: 161.
[0469] In some of any embodiments, said polynucleotide contains a
nucleic acid encoding the sequence set forth in SEQ ID NO: 184,
185, 186, 187, 188 or 189 or a sequence that encodes a polypeptide
sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the sequence set forth in SEQ ID NO: 184, 185,
186, 187, 188 or 189. In some of any embodiments, said
polynucleotide contains a nucleic acid encoding the sequence set
forth in SEQ ID NO: 184 or a sequence that encodes a polypeptide
sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the sequence set forth in SEQ ID NO: 184. In
some of any embodiments, said polynucleotide contains a nucleic
acid encoding the sequence set forth in SEQ ID NO: 184. In some of
any embodiments, said polynucleotide contains a nucleic acid
encoding the sequence set forth in SEQ ID NO: 185 or a sequence
that encodes a polypeptide sequence that exhibits at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence
set forth in SEQ ID NO: 185. In some of any embodiments, said
polynucleotide contains a nucleic acid encoding the sequence set
forth in SEQ ID NO: 185. In some of any embodiments, said
polynucleotide contains a nucleic acid encoding the sequence set
forth in SEQ ID NO: 186 or a sequence that encodes a polypeptide
sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the sequence set forth in SEQ ID NO: 186. In
some of any embodiments, said polynucleotide contains a nucleic
acid encoding the sequence set forth in SEQ ID NO: 186. In some of
any embodiments, said polynucleotide contains a nucleic acid
encoding the sequence set forth in SEQ ID NO: 187 or a sequence
that encodes a polypeptide sequence that exhibits at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence
set forth in SEQ ID NO: 187. In some of any embodiments, said
polynucleotide contains a nucleic acid encoding the sequence set
forth in SEQ ID NO: 187. In some of any embodiments, said
polynucleotide contains a nucleic acid encoding the sequence set
forth in SEQ ID NO: 188 or a sequence that encodes a polypeptide
sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the sequence set forth in SEQ ID NO: 188. In
some of any embodiments, said polynucleotide contains a nucleic
acid encoding the sequence set forth in SEQ ID NO: 188. In some of
any embodiments, said polynucleotide contains a nucleic acid
encoding the sequence set forth in SEQ ID NO: 189 or a sequence
that encodes a polypeptide sequence that exhibits at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence
set forth in SEQ ID NO: 189. In some of any embodiments, said
polynucleotide contains a nucleic acid encoding the sequence set
forth in SEQ ID NO: 189.
TABLE-US-00003 TABLE 3 Exemplary ROR1-binding CAR components (SEQ
ID NOS:) Nucleotides (nt) scFv spacer TM 4-1BB CD3.xi. CAR CAR #
scFv# N/O O/SSE N/O O/SSE N/O O/SSE N/O O/SSE N/O O/SSE O/SSE F
ROR1-1 116 117 192 136 197 198 154 155 182 150 156 A ROR1-2 125 126
192 136 197 198 154 155 182 150 157 G ROR1-1 116 117 193 139 197
198 154 155 182 150 158 I ROR1-3 107 108 193 139 197 198 154 155
182 150 159 R ROR1-2 125 126 193 139 197 198 154 155 182 150 160 B1
ROR1-4 132 133 193 139 197 198 154 155 182 150 161 Amino acids (aa)
CAR # scFv# scFv spacer TM 4-1BB CD3z CAR F ROR1-1 118 135 8 12 13
184 A ROR1-2 127 135 8 12 13 185 G ROR1-1 118 138 8 12 13 186 I
ROR1-3 109 138 8 12 13 187 R ROR1-2 127 138 8 12 13 188 B1 ROR1-4
134 138 8 12 13 189
II. ENGINEERED CELLS
[0470] Also provided are cells such as engineered cells that
contain a recombinant receptor (e.g., a chimeric antigen receptor)
such as one that contains an extracellular domain including an
anti-ROR1 antibody or fragment as described herein. Also provided
are populations of such cells, compositions containing such cells
and/or enriched for such cells, such as in which cells expressing
the ROR1-binding molecule make up at least 50, 60, 70, 80, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or more percent of the total cells
in the composition or cells of a certain type such as T cells or
CD8+ or CD4+ cells. Among the compositions are pharmaceutical
compositions and formulations for administration, such as for
adoptive cell therapy. Also provided are therapeutic methods for
administering the cells and compositions to subjects, e.g.,
patients.
[0471] Thus also provided are genetically engineered cells
expressing the recombinant receptors containing the antibodies,
e.g., cells containing the CARs. The cells generally are eukaryotic
cells, such as mammalian cells, and typically are human cells. In
some embodiments, the cells are derived from the blood, bone
marrow, lymph, or lymphoid organs, are cells of the immune system,
such as cells of the innate or adaptive immunity, e.g., myeloid or
lymphoid cells, including lymphocytes, typically T cells and/or NK
cells. Other exemplary cells include stem cells, such as
multipotent and pluripotent stem cells, including induced
pluripotent stem cells (iPSCs). The cells typically are primary
cells, such as those isolated directly from a subject and/or
isolated from a subject and frozen. In some embodiments, the cells
include one or more subsets of T cells or other cell types, such as
whole T cell populations, CD4.sup.+ cells, CD8.sup.+ cells, and
subpopulations thereof, such as those defined by function,
activation state, maturity, potential for differentiation,
expansion, recirculation, localization, and/or persistence
capacities, antigen-specificity, type of antigen receptor, presence
in a particular organ or compartment, marker or cytokine secretion
profile, and/or degree of differentiation. With reference to the
subject to be treated, the cells may be allogeneic and/or
autologous. Among the methods include off-the-shelf methods. In
some aspects, such as for off-the-shelf technologies, the cells are
pluripotent and/or multipotent, such as stem cells, such as induced
pluripotent stem cells (iPSCs). In some embodiments, the methods
include isolating cells from the subject, preparing, processing,
culturing, and/or engineering them, as described herein, and
re-introducing them into the same patient, before or after
cryopreservation.
[0472] Among the sub-types and subpopulations of T cells and/or of
CD4.sup.+ and/or of CD8.sup.+ T cells are naive T (T.sub.N) cells,
effector T cells (T.sub.EFF), memory T cells and sub-types thereof,
such as stem cell memory T (T.sub.SCM), central memory T
(T.sub.CM), effector memory T (T.sub.EM), or terminally
differentiated effector memory T cells, tumor-infiltrating
lymphocytes (TIL), immature T cells, mature T cells, helper T
cells, cytotoxic T cells, mucosa-associated invariant T (MAIT)
cells, naturally occurring and adaptive regulatory T (Treg) cells,
helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17
cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta
T cells, and delta/gamma T cells.
[0473] In some embodiments, the cells are natural killer (NK)
cells. In some embodiments, the cells are monocytes or
granulocytes, e.g., myeloid cells, macrophages, neutrophils,
dendritic cells, mast cells, eosinophils, and/or basophils.
[0474] In some embodiments, the cells include one or more
polynucleotides introduced via genetic engineering, and thereby
express recombinant or genetically engineered products of such
polynucleotides. In some embodiments, the polynucleotides are
heterologous, i.e., normally not present in a cell or sample
obtained from the cell, such as one obtained from another organism
or cell, which for example, is not ordinarily found in the cell
being engineered and/or an organism from which such cell is
derived. In some embodiments, the polynucleotides are not naturally
occurring, such as a polynucleotide not found in nature, including
one comprising chimeric combinations of polynucleotides encoding
various domains from multiple different cell types. In some
embodiments, the cells (e.g., engineered cells) comprise a vector
(e.g., a viral vector, expression vector, etc.) as described herein
such as a vector comprising a nucleic acid encoding a recombinant
receptor described herein.
[0475] A. Vectors and Methods for Genetic Engineering
[0476] Also provided are nucleic acids, e.g., polynucleotides,
encoding the antibodies and/or portions, e.g., chains, thereof.
Among the provided nucleic acids are those encoding the anti-ROR1
antibodies (e.g., antigen-binding fragment) described herein. Also
provided are nucleic acids, e.g., polynucleotides, encoding one or
more antibodies and/or portions thereof, e.g., those encoding one
or more of the anti-ROR1 antibodies (e.g., antigen-binding
fragment) described herein and/or other antibodies and/or portions
thereof, e.g., antibodies and/or portions thereof that binds other
target antigens. Also provided are methods, nucleic acids,
compositions, and kits, for expressing the binding molecules (e.g.,
anti-ROR1 binding molecules), including recombinant receptors
(e.g., CARs) comprising the binding molecules, and for producing
the genetically engineered cells expressing such binding molecules.
In some embodiments, one or more binding molecules, including
recombinant receptors (e.g., CARs) can be genetically engineered
into cells or plurality of cells. The genetic engineering generally
involves introduction of a nucleic acid encoding the recombinant or
engineered component into the cell, such as by retroviral
transduction, transfection, or transformation.
[0477] The nucleic acids may include those encompassing natural
and/or non-naturally occurring nucleotides and bases, e.g.,
including those with backbone modifications. The terms "nucleic
acid molecule", "nucleic acid" and "polynucleotide" may be used
interchangeably, and refer to a polymer of nucleotides. Such
polymers of nucleotides may contain natural and/or non-natural
nucleotides, and include, but are not limited to, DNA, RNA, and
PNA. "Nucleic acid sequence" refers to the linear sequence of
nucleotides that comprise the nucleic acid molecule or
polynucleotide. In some aspects, the nucleic acid sequence encoding
at least a portion of the antibody or antigen-binding fragment
thereof, such as an scFv, conjugates, receptors (e.g., CARs)
provided herein can be optimized, for example, codon-optimized for
expression in a human cell and/or optimized to reduce or eliminate
cryptic splice sites.
[0478] In some embodiments, the polynucleotides also include one or
more additional sequences, such as those encoding one or more
additional molecules, such as a marker, or promoters, regulatory
elements and/or multicistronic elements. In some embodiments, the
provided polynucleotides include any of the polynucleotides
described herein, e.g., in Section I.E.
[0479] Also provided are vectors containing the nucleic acids,
e.g., polynucleotides, and host cells containing the vectors, e.g.,
for producing the antibodies or antigen-binding fragments thereof.
Also provided are methods for producing the antibodies or
antigen-binding fragments thereof. The nucleic acid may encode an
amino acid sequence comprising the V.sub.L region and/or an amino
acid sequence comprising the V.sub.H region of the antibody (e.g.,
the light and/or heavy chains of the antibody). The nucleic acid
may encode one or more amino acid sequence comprising the V.sub.L
region and/or an amino acid sequence comprising the V.sub.H region
of the antibody (e.g., the light and/or heavy chains of the
antibody). In some embodiments, the nucleic acid, e.g.,
polynucleotide encodes one or more V.sub.H region and/or one or
more V.sub.L region of the antibody, in any order or orientation.
In some embodiments, the nucleic acid, e.g., polynucleotide encodes
a V.sub.H region and a V.sub.L region, and the coding sequence for
the V.sub.H region is upstream of the coding sequence for the
V.sub.L region. In some embodiments, the nucleic acid, e.g.,
polynucleotide encodes a V.sub.H region and a V.sub.L region, and
the coding sequence for the V.sub.L region is upstream of the
coding sequence for the V.sub.H region.
[0480] Also provided are vectors containing the nucleic acids,
e.g., polynucleotides, and engineered cells containing the vectors,
e.g., engineered immune cells expressing the binding molecules such
as recombinant receptors. Also provided are methods for engineering
cells, such as immune cells, to express the anti-ROR1 binding
molecule, such as a recombinant receptor, e.g., a chimeric antigen
receptor (CAR). The nucleic acid may encode an amino acid sequence
comprising the V.sub.L region and/or an amino acid sequence
comprising the V.sub.H region of the antibody (e.g., the light
and/or heavy chains of the antibody) as the extracellular
antigen-binding domain; a transmembrane domain and intracellular
domains, such as a CD3 zeta and a costimulatory signaling
domain
[0481] In a further embodiment, one or more vectors (e.g.,
expression vectors) comprising such polynucleotides are provided.
In a further embodiment, a host cell comprising such
polynucleotides is provided. In one such embodiment, a host cell
comprises (e.g., has been transformed with) a vector comprising a
nucleic acid that encodes an amino acid sequence comprising the
V.sub.H region of the antibody. In another such embodiment, a host
cell comprises (e.g., has been transformed with) (1) a vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the V.sub.L region of the antibody and an amino acid
sequence comprising the V.sub.H region of the antibody, or (2) a
first vector comprising a nucleic acid that encodes an amino acid
sequence comprising the V.sub.L region of the antibody and a second
vector comprising a nucleic acid that encodes an amino acid
sequence comprising the V.sub.H region of the antibody. In some
embodiments, a host cell comprises (e.g., has been transformed
with) one or more vectors comprising one or more nucleic acid that
encodes one or more an amino acid sequence comprising one or more
antibodies and/or portions thereof, e.g., antigen-binding fragments
thereof. In some embodiments, one or more such host cells are
provided. In some embodiments, a composition containing one or more
such host cells are provided. In some embodiments, the one or more
host cells can express different antibodies, or the same antibody.
In some embodiments, each of the host cells can express more than
one antibody.
[0482] Also provided are methods of making the anti-ROR1 chimeric
antigen receptors. For recombinant production of the chimeric
receptors, a nucleic acid sequence encoding a chimeric receptor
antibody, e.g., as described herein, may be isolated and inserted
into one or more vectors for further cloning and/or expression in a
host cell. Such nucleic acid sequences may be readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the antibody). In some
embodiments, a method of making the anti-ROR1 chimeric antigen
receptor is provided, wherein the method comprises culturing a host
cell comprising a nucleic acid sequence encoding the antibody, as
provided above, under conditions suitable for expression of the
receptor.
[0483] Also provided are methods of making the anti-ROR1 antibodies
(including antigen-binding fragments). For recombinant production
of the anti-ROR1 antibody, a nucleic acid sequence or a
polynucleotide encoding an antibody, e.g., as described above, may
be isolated and inserted into one or more vectors for further
cloning and/or expression in a host cell. Such nucleic acid
sequences may be readily isolated and sequenced using conventional
procedures (e.g., by using oligonucleotide probes that are capable
of binding specifically to genes encoding the heavy and light
chains of the antibody). In some embodiments, a method of making
the anti-ROR1 antibody is provided, wherein the method comprises
culturing a host cell comprising a nucleic acid sequence encoding
the antibody, as provided above, under conditions suitable for
expression of the antibody, and optionally recovering the antibody
from the host cell (or host cell culture medium).
[0484] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been modified to mimic or
approximate those in human cells, resulting in the production of an
antibody with a partially or fully human glycosylation pattern. See
Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat.
Biotech. 24:210-215 (2006).
[0485] Exemplary eukaryotic cells that may be used to express
polypeptides include, but are not limited to, COS cells, including
COS 7 cells; 293 cells, including 293-6E cells; CHO cells,
including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells;
PER.C6.RTM. cells; and NSO cells. In some embodiments, the antibody
heavy chains and/or light chains (e.g., V.sub.H region and/or
V.sub.L region) may be expressed in yeast. See, e.g., U.S.
Publication No. US 2006/0270045 A1. In some embodiments, a
particular eukaryotic host cell is selected based on its ability to
make desired post-translational modifications to the heavy chains
and/or light chains (e.g., V.sub.H region and/or V.sub.L region).
For example, in some embodiments, CHO cells produce polypeptides
that have a higher level of sialylation than the same polypeptide
produced in 293 cells.
[0486] In some embodiments, the antibody or antigen-binding
fragment provided herein is produced in a cell-free system.
Exemplary cell-free systems are described, e.g., in Sitaraman et
al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends
Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21:
695-713 (2003).
[0487] The provided embodiments further include vectors and host
cells and other expression systems for expressing and producing the
antibodies and other antigen-binding proteins, including eukaryotic
and prokaryotic host cells, including bacteria, filamentous fungi,
and yeast, as well as mammalian cells such as human cells, as well
as cell-free expression systems.
[0488] In some embodiments, gene transfer is accomplished by first
stimulating the cell, such as by combining it with a stimulus that
induces a response such as proliferation, survival, and/or
activation, e.g., as measured by expression of a cytokine or
activation marker, followed by transduction of the activated cells,
and expansion in culture to numbers sufficient for clinical
applications.
[0489] In some contexts, overexpression of a stimulatory factor
(for example, a lymphokine or a cytokine) may be toxic to a
subject. Thus, in some contexts, the engineered cells include gene
segments that cause the cells to be susceptible to negative
selection in vivo, such as upon administration in adoptive
immunotherapy. For example in some aspects, the cells are
engineered so that they can be eliminated as a result of a change
in the in vivo condition of the patient to which they are
administered. The negative selectable phenotype may result from the
insertion of a gene that confers sensitivity to an administered
agent, for example, a compound. Negative selectable genes include
the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene
(Wigler et al., Cell 2:223, 1977) which confers ganciclovir
sensitivity; the cellular hypoxanthine phosphoribosyltransferase
(HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT)
gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl.
Acad. Sci. USA. 89:33 (1992)).
[0490] In some aspects, the cells further are engineered to promote
expression of cytokines or other factors. Various methods for the
introduction of genetically engineered components, e.g., antigen
receptors, e.g., CARs, are well known and may be used with the
provided methods and compositions. Exemplary methods include those
for transfer of polynucleotides encoding the receptors, including
via viral, e.g., retroviral or lentiviral, transduction,
transposons, and electroporation.
[0491] In some embodiments, recombinant polynucleotides are
transferred into cells using recombinant infectious virus
particles, such as, e.g., vectors derived from simian virus 40
(SV40), adenoviruses, adeno-associated virus (AAV). In some
embodiments, recombinant polynucleotides are transferred into T
cells using recombinant lentiviral vectors, such as HIV-1
lentivirus-based vectors (lentivectors; see, e.g., Amado et al.,
Science. 1999 Jul. 30; 285(5428):674-676), or retroviral vectors,
such as gamma-retroviral vectors (see, e.g., Koste et al. (2014)
Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al.
(2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol
Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 Nov.
29(11): 550-557).
[0492] In some embodiments, the retroviral vector or lentiviral
vector has a long terminal repeat sequence (LTR). In some
embodiments the vector is derived from the Moloney murine leukemia
virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine
embryonic stem cell virus (MESV), murine stem cell virus (MSCV),
spleen focus forming virus (SFFV), human immunodeficiency virus
type 1 (HIV-1) or human immunodeficiency virus type 2 (HIV-2/SIV).
In some embodiments, the vectors are self-inactivating (SIN). In
some embodiments, the vectors are conditionally replicating
(mobilizable) vectors. Most lentiviral vectors are derived from
human, feline or simian lentiviruses. Most retroviral vectors are
derived from murine retroviruses. In some embodiments, the
lentiviruses or retroviruses include those derived from any avian
or mammalian cell source. The lentiviruses or retroviruses
typically are amphotropic, meaning that they are capable of
infecting host cells of several species, including humans. In one
embodiment, the gene to be expressed replaces the retroviral gag,
pol and/or env sequences. Methods of lentiviral transduction are
known. Exemplary methods are described in, e.g., Wang et al. (2012)
J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood.
101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506:
97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505. A
number of illustrative retroviral systems have also been described
(e.g., Amado et al., (1999) Science 285(5428):674-676, U.S. Pat.
Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989)
BioTechniques 7:980-990; Miller (1990) Human Gene Therapy 1:5-14;
Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993)
Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin
(1993) Cur. Opin. Genet. Develop. 3:102-109).
[0493] In some embodiments, recombinant polynucleotides are
transferred into T cells via electroporation (see, e.g., Chicaybam
et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000)
Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant
polynucleotides are transferred into T cells via transposition
(see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437;
Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al.
(2009) Methods Mol Biol 506: 115-126). Other methods of introducing
and expressing genetic material in immune cells include calcium
phosphate transfection (e.g., as described in Current Protocols in
Molecular Biology, John Wiley & Sons, New York. N.Y.),
protoplast fusion, cationic liposome-mediated transfection;
tungsten particle-facilitated microparticle bombardment (Johnston
(1990) Nature 346: 776-777); and strontium phosphate DNA
co-precipitation (Brash et al., (1987) Mol. Cell Biol. 7:
2031-2034). Other approaches and vectors for transfer of the
polynucleotides encoding the recombinant products are those
described, e.g., in WO2014055668, and U.S. Pat. No. 7,446,190.
[0494] Among additional polynucleotides, e.g., genes for
introduction are those to improve the outcome of therapy, such as
by promoting viability and/or function of transferred cells; genes
to provide a genetic marker for selection and/or evaluation of the
cells, such as to assess in vivo survival or localization; genes to
improve safety, for example, by making the cell susceptible to
negative selection in vivo as described by Lupton S. D. et al.,
Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene
Therapy 3:319-338 (1992); see also the publications of
PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the
use of bifunctional selectable fusion genes derived from fusing a
dominant positive selectable marker with a negative selectable
marker. See, e.g., Riddell et al., U.S. Pat. No. 6,040,177, at
columns 14-17.
[0495] In some embodiments the vector or construct can contain a
promoter and/or enhancer or regulatory elements to regulate
expression of the encoded recombinant receptor. In some examples
the promoter and/or enhancer or regulatory elements can be
condition-dependent promoters, enhancers, and/or regulatory
elements. In some examples these elements drive expression of the
transgene. In some examples, the CAR transgene can be operatively
linked to a promoter, such as an EF1alpha promoter with an HTLV1
enhancer (SEQ ID NO:164). In some examples, the CAR transgene is
operatively linked to a Woodchuck Hepatitis Virus (WHP)
Posttranscriptional Regulatory Element (WPRE; SEQ ID NO: 165),
located downstream of the transgene.
[0496] In some embodiments, the vector or construct can contain a
single promoter that drives the expression of one or more nucleic
acid molecules. In some embodiments, such nucleic acid molecules,
e.g., transcripts, can be multicistronic (bicistronic or
tricistronic, see e.g., U.S. Pat. No. 6,060,273). For example, in
some embodiments, transcription units can be engineered as a
bicistronic unit containing an IRES (internal ribosome entry site),
which allows coexpression of gene products (e.g., encoding a first
and second chimeric receptor) by a message from a single promoter.
For example, in some embodiments, the vector or construct can
contain a nucleic acid encoding an anti-ROR1 receptor (e.g., an
anti-ROR1 CAR) provided herein and a nucleic acid encoding a
different molecule, separated by an IRES, under the regulation of a
single promoter.
[0497] Alternatively, in some cases, a single promoter may direct
expression of an RNA that contains, in a single open reading frame
(ORF), two or three genes (e.g. encoding a first and second binding
molecules, e.g., antibody recombinant receptor) separated from one
another by sequences encoding a self-cleavage peptide (e.g., 2A
cleavage sequences) or a protease recognition site (e.g., furin).
The ORF thus encodes a single polypeptide, which, either during (in
the case of T2A) or after translation, is cleaved into the
individual proteins. In some cases, the peptide, such as T2A, can
cause the ribosome to skip (ribosome skipping) synthesis of a
peptide bond at the C-terminus of a 2A element, leading to
separation between the end of the 2A sequence and the next peptide
downstream (see, for example, de Felipe. Genetic Vaccines and Ther.
2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)). Many 2A
elements are known. Examples of 2A sequences that can be used in
the methods and polynucleotides disclosed herein, without
limitation, 2A sequences from the foot-and-mouth disease virus
(F2A, e.g., SEQ ID NO:21 or 168), equine rhinitis A virus (E2A,
e.g., SEQ ID NO: 20 or 167), Thosea asigna virus (T2A, e.g., SEQ ID
NO: 6, 17 or 166), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO:
18 or 19) as described in U.S. Patent Publication No. 20070116690.
In some embodiments, the one or more different or separate
promoters drive the expression of one or more nucleic acid
molecules encoding the one or more binding molecules, e.g.,
recombinant receptors.
[0498] Any of the recombinant receptors provided herein, e.g.,
anti-ROR1 recombinant receptors and/or the additional recombinant
receptors, can be encoded by polynucleotides containing one or more
nucleic acid molecules encoding the receptors, in any combinations
or arrangements. For example, one, two, three or more
polynucleotides can encode one, two, three or more different
receptors or domains. In some embodiments, one vector or construct
contains nucleic acid molecules encoding one or more recombinant
receptor(s), and a separate vector or construct contains nucleic
acid molecules encoding an additional binding molecule, e.g.,
antibody and/or recombinant receptor, such as an anti-ROR1 receptor
(e.g., anti-ROR1 CAR).
[0499] In some embodiments, the nucleic acid molecules can also
encode one or more surrogate marker(s), such as fluorescent protein
(e.g., green fluorescent protein (GFP)) or a cell surface marker
(e.g., a truncated surface marker such as truncated EGFR (tEGFR),
which may be used to confirm transduction or engineering of the
cell to express the receptor. For example, in some aspects,
extrinsic marker genes are utilized in connection with engineered
cell therapies to permit detection or selection of cells and, in
some cases, also to promote cell suicide by ADCC. Exemplary marker
genes include truncated epidermal growth factor receptor (EGFRt),
which can be co-expressed with a transgene of interest (e.g., a CAR
or TCR) in transduced cells (see, e.g., U.S. Pat. No. 8,802,374).
EGFRt contains an epitope recognized by the antibody cetuximab
(Erbitux.RTM.). For this reason, Erbitux.RTM. can be used to
identify or select cells that have been engineered with the EGFRt
construct, including in cells also co-engineered with another
recombinant receptor, such as a chimeric antigen receptor
(CAR).
[0500] In some embodiments, the nucleic acid encoding the binding
molecules further contain contains a nucleic acid sequence encoding
one or more marker(s). In some embodiments, the one or more
marker(s) is a transduction marker, surrogate marker and/or a
selection marker.
[0501] In some embodiments, the marker is a transduction marker or
a surrogate marker. A transduction marker or a surrogate marker can
be used to detect cells that have been introduced with the
polynucleotide, e.g., a polynucleotide encoding a recombinant
receptor. In some embodiments, the transduction marker can indicate
or confirm modification of a cell. In some embodiments, the
surrogate marker is a protein that is made to be co-expressed on
the cell surface with the recombinant receptor, e.g. CAR. In
particular embodiments, such a surrogate marker is a surface
protein that has been modified to have little or no activity. In
certain embodiments, the surrogate marker is encoded on the same
polynucleotide that encodes the recombinant receptor. In some
embodiments, the nucleic acid sequence encoding the recombinant
receptor is operably linked to a nucleic acid sequence encoding a
marker, optionally separated by an internal ribosome entry site
(IRES), or a nucleic acid encoding a self-cleaving peptide or a
peptide that causes ribosome skipping, such as a 2A sequence, such
as a T2A, a P2A, an E2A or an F2A. Extrinsic marker genes may in
some cases be utilized in connection with engineered cell to permit
detection or selection of cells and, in some cases, also to promote
cell suicide.
[0502] Exemplary surrogate markers can include truncated forms of
cell surface polypeptides, such as truncated forms that are
non-functional and to not transduce or are not capable of
transducing a signal or a signal ordinarily transduced by the
full-length form of the cell surface polypeptide, and/or do not or
are not capable of internalizing Exemplary truncated cell surface
polypeptides including truncated forms of growth factors or other
receptors such as a truncated human epidermal growth factor
receptor 2 (tHER2), a truncated epidermal growth factor receptor
(tEGFR, exemplary tEGFR sequence set forth in SEQ ID NO:7, 16 or
153) or a prostate-specific membrane antigen (PSMA) or modified
form thereof. tEGFR may contain an epitope recognized by the
antibody cetuximab (Erbitux.RTM.) or other therapeutic anti-EGFR
antibody or binding molecule, which can be used to identify or
select cells that have been engineered with the tEGFR construct and
an encoded exogenous protein, and/or to eliminate or separate cells
expressing the encoded exogenous protein. See U.S. Pat. No.
8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4):
430-434). In some aspects, the marker, e.g. surrogate marker,
includes all or part (e.g., truncated form) of CD34, a NGFR, a CD19
or a truncated CD19, e.g., a truncated non-human CD19, or epidermal
growth factor receptor (e.g., tEGFR).
[0503] In some embodiments, the marker is or comprises a
fluorescent protein, such as green fluorescent protein (GFP),
enhanced green fluorescent protein (EGFP), such as super-fold GFP
(sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry,
mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein
(CFP), blue green fluorescent protein (BFP), enhanced blue
fluorescent protein (EBFP), and yellow fluorescent protein (YFP),
and variants thereof, including species variants, monomeric
variants, and codon-optimized and/or enhanced variants of the
fluorescent proteins. In some embodiments, the marker is or
comprises an enzyme, such as a luciferase, the lacZ gene from E.
coli, alkaline phosphatase, secreted embryonic alkaline phosphatase
(SEAP), chloramphenicol acetyl transferase (CAT). Exemplary
light-emitting reporter genes include luciferase (luc),
.beta.-galactosidase, chloramphenicol acetyltransferase (CAT),
.beta.-glucuronidase (GUS) or variants thereof.
[0504] In some embodiments, the marker is a selection marker. In
some embodiments, the selection marker is or comprises a
polypeptide that confers resistance to exogenous agents or drugs.
In some embodiments, the selection marker is an antibiotic
resistance gene. In some embodiments, the selection marker is an
antibiotic resistance gene confers antibiotic resistance to a
mammalian cell. In some embodiments, the selection marker is or
comprises a Puromycin resistance gene, a Hygromycin resistance
gene, a Blasticidin resistance gene, a Neomycin resistance gene, a
Geneticin resistance gene or a Zeocin resistance gene or a modified
form thereof.
[0505] In some embodiments, the nucleic acid encoding the marker is
operably linked to a polynucleotide encoding for a linker sequence,
such as a cleavable linker sequence, e.g., a T2A. For example, a
marker, and optionally a linker sequence, can be any as disclosed
in PCT Pub. No. WO2014031687. For example, the marker can be a
truncated EGFR (tEGFR) that is, optionally, linked to a linker
sequence, such as a T2A cleavable linker sequence. An exemplary
polypeptide for a truncated EGFR (e.g. tEGFR) comprises the
sequence of amino acids set forth in SEQ ID NO: 7, 16 or 153 or a
sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity to SEQ ID NO: 7, 16 or 153.
[0506] In some embodiments, the marker is a molecule, e.g., cell
surface protein, not naturally found on T cells or not naturally
found on the surface of T cells, or a portion thereof.
[0507] In some embodiments, the molecule is a non-self molecule,
e.g., non-self protein, i.e., one that is not recognized as "self"
by the immune system of the host into which the cells will be
adoptively transferred.
[0508] In some embodiments, the marker serves no therapeutic
function and/or produces no effect other than to be used as a
marker for genetic engineering, e.g., for selecting cells
successfully engineered. In other embodiments, the marker may be a
therapeutic molecule or molecule otherwise exerting some desired
effect, such as a ligand for a cell to be encountered in vivo, such
as a costimulatory or immune checkpoint molecule to enhance and/or
dampen responses of the cells upon adoptive transfer and encounter
with ligand.
[0509] Also provided are compositions containing one or more of the
nucleic acid molecules, vectors or constructs, such as any
described above. In some embodiments, the nucleic acid molecules,
vectors, constructs or compositions can be used to engineer cells,
such as T cells, to express any of the binding molecules, e.g.,
antibody or recombinant receptor, and/or the additional binding
molecules.
[0510] B. Preparation of Cells for Engineering
[0511] In some embodiments, preparation of the engineered cells
includes one or more culture and/or preparation steps. The cells
for introduction of the recombinant receptor (e.g., CAR) may be
isolated from a sample, such as a biological sample, e.g., one
obtained from or derived from a subject. In some embodiments, the
subject from which the cell is isolated is one having the disease
or condition or in need of a cell therapy or to which cell therapy
will be administered. The subject in some embodiments is a human in
need of a particular therapeutic intervention, such as the adoptive
cell therapy for which cells are being isolated, processed, and/or
engineered.
[0512] Accordingly, the cells in some embodiments are primary
cells, e.g., primary human cells. The samples include tissue,
fluid, and other samples taken directly from the subject, as well
as samples resulting from one or more processing steps, such as
separation, centrifugation, genetic engineering (e.g., transduction
with viral vector), washing, and/or incubation. The biological
sample can be a sample obtained directly from a biological source
or a sample that is processed. Biological samples include, but are
not limited to, body fluids, such as blood, plasma, serum,
cerebrospinal fluid, synovial fluid, urine and sweat, tissue and
organ samples, including processed samples derived therefrom.
[0513] In some aspects, the sample from which the cells are derived
or isolated is blood or a blood-derived sample, or is or is derived
from an apheresis or leukapheresis product. Exemplary samples
include whole blood, peripheral blood mononuclear cells (PBMCs),
leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia,
lymphoma, lymph node, gut associated lymphoid tissue, mucosa
associated lymphoid tissue, spleen, other lymphoid tissues, liver,
lung, stomach, intestine, colon, kidney, pancreas, breast, bone,
prostate, cervix, testes, ovaries, tonsil, or other organ, and/or
cells derived therefrom. Samples include, in the context of cell
therapy, e.g., adoptive cell therapy, samples from autologous and
allogeneic sources.
[0514] In some embodiments, the cells are derived from cell lines,
e.g., T cell lines. The cells in some embodiments are obtained from
a xenogeneic source, for example, from mouse, rat, non-human
primate, or pig.
[0515] In some embodiments, isolation of the cells includes one or
more preparation and/or non-affinity based cell separation steps.
In some examples, cells are washed, centrifuged, and/or incubated
in the presence of one or more reagents, for example, to remove
unwanted components, enrich for desired components, lyse or remove
cells sensitive to particular reagents. In some examples, cells are
separated based on one or more property, such as density, adherent
properties, size, sensitivity and/or resistance to particular
components.
[0516] In some examples, cells from the circulating blood of a
subject are obtained, e.g., by apheresis or leukapheresis. The
samples, in some aspects, contain lymphocytes, including T cells,
monocytes, granulocytes, B cells, other nucleated white blood
cells, red blood cells, and/or platelets, and in some aspects
contain cells other than red blood cells and platelets.
[0517] In some embodiments, the blood cells collected from the
subject are washed, e.g., to remove the plasma fraction and to
place the cells in an appropriate buffer or media for subsequent
processing steps. In some embodiments, the cells are washed with
phosphate buffered saline (PBS). In some embodiments, the wash
solution lacks calcium and/or magnesium and/or many or all divalent
cations. In some aspects, a washing step is accomplished a
semi-automated "flow-through" centrifuge (for example, the Cobe
2991 cell processor, Baxter) according to the manufacturer's
instructions. In some aspects, a washing step is accomplished by
tangential flow filtration (TFF) according to the manufacturer's
instructions. In some embodiments, the cells are resuspended in a
variety of biocompatible buffers after washing, such as, for
example, Ca++/Mg++ free PBS. In certain embodiments, components of
a blood cell sample are removed and the cells directly resuspended
in culture media.
[0518] In some embodiments, the methods include density-based cell
separation methods, such as the preparation of white blood cells
from peripheral blood by lysing the red blood cells and
centrifugation through a Percoll or Ficoll gradient.
[0519] In some embodiments, the isolation methods include the
separation of different cell types based on the expression or
presence in the cell of one or more specific molecules, such as
surface markers, e.g., surface proteins, intracellular markers, or
nucleic acid. In some embodiments, any known method for separation
based on such markers may be used. In some embodiments, the
separation is affinity- or immunoaffinity-based separation. For
example, the isolation in some aspects includes separation of cells
and cell populations based on the cells' expression or expression
level of one or more markers, typically cell surface markers, for
example, by incubation with an antibody or binding partner that
specifically binds to such markers, followed generally by washing
steps and separation of cells having bound the antibody or binding
partner, from those cells having not bound to the antibody or
binding partner.
[0520] Such separation steps can be based on positive selection, in
which the cells having bound the reagents are retained for further
use, and/or negative selection, in which the cells having not bound
to the antibody or binding partner are retained. In some examples,
both fractions are retained for further use. In some aspects,
negative selection can be particularly useful where no antibody is
available that specifically identifies a cell type in a
heterogeneous population, such that separation is best carried out
based on markers expressed by cells other than the desired
population.
[0521] The separation need not result in 100% enrichment or removal
of a particular cell population or cells expressing a particular
marker. For example, positive selection of or enrichment for cells
of a particular type, such as those expressing a marker, refers to
increasing the number or percentage of such cells, but need not
result in a complete absence of cells not expressing the marker.
Likewise, negative selection, removal, or depletion of cells of a
particular type, such as those expressing a marker, refers to
decreasing the number or percentage of such cells, but need not
result in a complete removal of all such cells.
[0522] In some examples, multiple rounds of separation steps are
carried out, where the positively or negatively selected fraction
from one step is subjected to another separation step, such as a
subsequent positive or negative selection. In some examples, a
single separation step can deplete cells expressing multiple
markers simultaneously, such as by incubating cells with a
plurality of antibodies or binding partners, each specific for a
marker targeted for negative selection. Likewise, multiple cell
types can simultaneously be positively selected by incubating cells
with a plurality of antibodies or binding partners expressed on the
various cell types.
[0523] For example, in some aspects, specific subpopulations of T
cells, such as cells positive or expressing high levels of one or
more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+,
CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by
positive or negative selection techniques.
[0524] For example, CD3+, CD28+ T cells can be positively selected
using anti-CD3/anti-CD28 conjugated magnetic beads (e.g.,
DYNABEADS.RTM. M-450 CD3/CD28 T Cell Expander, MACSiBeads.TM.,
etc.).
[0525] In some embodiments, isolation is carried out by enrichment
for a particular cell population by positive selection, or
depletion of a particular cell population, by negative selection.
In some embodiments, positive or negative selection is accomplished
by incubating cells with one or more antibodies or other binding
agent that specifically bind to one or more surface markers
expressed or expressed (marker+) at a relatively higher level
(marker.sup.high) on the positively or negatively selected cells,
respectively.
[0526] In some embodiments, T cells are separated from a PBMC
sample by negative selection of markers expressed on non-T cells,
such as B cells, monocytes, or other white blood cells, such as
CD14. In some aspects, a CD4+ or CD8+ selection step is used to
separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+
populations can be further sorted into sub-populations by positive
or negative selection for markers expressed or expressed to a
relatively higher degree on one or more naive, memory, and/or
effector T cell subpopulations.
[0527] In some embodiments, CD8+ cells are further enriched for or
depleted of naive, central memory, effector memory, and/or central
memory stem cells, such as by positive or negative selection based
on surface antigens associated with the respective subpopulation.
In some embodiments, enrichment for central memory T (T.sub.CM)
cells is carried out to increase certain features, such as to
improve long-term survival, expansion, and/or engraftment following
administration, which in some aspects is particularly robust in
such sub-populations (see Terakura et al. (2012) Blood.1:72-82;
Wang et al. (2012) J Immunother. 35(9):689-701). In some
embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells
further enhances response.
[0528] In embodiments, memory T cells are present in both CD62L+
and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC can
be enriched for or depleted of CD62L-CD8+ and/or CD62L+CD8+
fractions, such as using anti-CD8 and anti-CD62L antibodies.
[0529] In some embodiments, the enrichment for central memory T
(T.sub.CM) cells is based on positive or high surface expression of
CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it
is based on negative selection for cells expressing or highly
expressing CD45RA and/or granzyme B. In some aspects, isolation of
a CD8+ population enriched for TCM cells is carried out by
depletion of cells expressing CD4, CD14, CD45RA, and positive
selection or enrichment for cells expressing CD62L. In one aspect,
enrichment for central memory T (T.sub.CM) cells is carried out
starting with a negative fraction of cells selected based on CD4
expression, which is subjected to a negative selection based on
expression of CD14 and CD45RA, and a positive selection based on
CD62L. Such selections in some aspects are carried out
simultaneously and in other aspects are carried out sequentially,
in either order. In some aspects, the same CD4 expression-based
selection step used in preparing the CD8+ cell population or
subpopulation, also is used to generate the CD4+ cell population or
sub-population, such that both the positive and negative fractions
from the CD4-based separation are retained and used in subsequent
steps of the methods, optionally following one or more further
positive or negative selection steps.
[0530] In a particular example, a sample of PBMCs or other white
blood cell sample is subjected to selection of CD4+ cells, where
both the negative and positive fractions are retained. The negative
fraction then is subjected to negative selection based on
expression of CD14 and CD45RA, and positive selection based on a
marker characteristic of central memory T cells, such as CD62L or
CCR7, where the positive and negative selections are carried out in
either order.
[0531] CD4+ T helper cells are sorted into naive, central memory,
and effector cells by identifying cell populations that have cell
surface antigens. CD4+ lymphocytes can be obtained by standard
methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-,
CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory
CD4.sup.+ cells are CD62L+ and CD45RO+. In some embodiments,
effector CD4+ cells are CD62L- and CD45RO-.
[0532] In one example, to enrich for CD4+ cells by negative
selection, a monoclonal antibody cocktail typically includes
antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some
embodiments, the antibody or binding partner is bound to a solid
support or matrix, such as a magnetic bead or paramagnetic bead, to
allow for separation of cells for positive and/or negative
selection. For example, in some embodiments, the cells and cell
populations are separated or isolated using immunomagnetic (or
affinity magnetic) separation techniques (reviewed in Methods in
Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2:
Cell Behavior In vitro and In vivo, p 17-25 Edited by: S. A. Brooks
and U. Schumacher.COPYRGT. Humana Press Inc., Totowa, N.J.).
[0533] In some aspects, the sample or composition of cells to be
separated is incubated with small, magnetizable or magnetically
responsive material, such as magnetically responsive particles or
microparticles, such as paramagnetic beads (e.g., such as
Dynabeads.RTM. or MACS.RTM. beads). The magnetically responsive
material, e.g., particle, generally is directly or indirectly
attached to a binding partner, e.g., an antibody, that specifically
binds to a molecule, e.g., surface marker, present on the cell,
cells, or population of cells that it is desired to separate, e.g.,
that it is desired to negatively or positively select.
[0534] In some embodiments, the magnetic particle or bead comprises
a magnetically responsive material bound to a specific binding
member, such as an antibody or other binding partner. There are
many well-known magnetically responsive materials used in magnetic
separation methods. Suitable magnetic particles include those
described in Molday, U.S. Pat. No. 4,452,773, and in European
Patent Specification EP 452342 B, which are hereby incorporated by
reference. Colloidal sized particles, such as those described in
Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No.
5,200,084, are other examples.
[0535] The incubation generally is carried out under conditions
whereby the antibodies or binding partners, or molecules, such as
secondary antibodies or other reagents, which specifically bind to
such antibodies or binding partners, which are attached to the
magnetic particle or bead, specifically bind to cell surface
molecules if present on cells within the sample.
[0536] In some aspects, the sample is placed in a magnetic field,
and those cells having magnetically responsive or magnetizable
particles attached thereto will be attracted to the magnet and
separated from the unlabeled cells. For positive selection, cells
that are attracted to the magnet are retained; for negative
selection, cells that are not attracted (unlabeled cells) are
retained. In some aspects, a combination of positive and negative
selection is performed during the same selection step, where the
positive and negative fractions are retained and further processed
or subject to further separation steps.
[0537] In certain embodiments, the magnetically responsive
particles are coated in primary antibodies or other binding
partners, secondary antibodies, lectins, enzymes, or streptavidin.
In certain embodiments, the magnetic particles are attached to
cells via a coating of primary antibodies specific for one or more
markers. In certain embodiments, the cells, rather than the beads,
are labeled with a primary antibody or binding partner, and then
cell-type specific secondary antibody- or other binding partner
(e.g., streptavidin)-coated magnetic particles, are added. In
certain embodiments, streptavidin-coated magnetic particles are
used in conjunction with biotinylated primary or secondary
antibodies.
[0538] In some embodiments, the magnetically responsive particles
are left attached to the cells that are to be subsequently
incubated, cultured and/or engineered; in some aspects, the
particles are left attached to the cells for administration to a
patient. In some embodiments, the magnetizable or magnetically
responsive particles are removed from the cells. Methods for
removing magnetizable particles from cells are known and include,
e.g., the use of competing non-labeled antibodies, magnetizable
particles or antibodies conjugated to cleavable linkers, etc. In
some embodiments, the magnetizable particles are biodegradable.
[0539] In some embodiments, the affinity-based selection is via
magnetic-activated cell sorting (MACS.RTM.) (Miltenyi Biotec,
Auburn, Calif.). Magnetic Activated Cell Sorting (MACS.RTM.)
systems are capable of high-purity selection of cells having
magnetized particles attached thereto. In certain embodiments,
MACS.RTM. operates in a mode wherein the non-target and target
species are sequentially eluted after the application of the
external magnetic field. That is, the cells attached to magnetized
particles are held in place while the unattached species are
eluted. Then, after this first elution step is completed, the
species that were trapped in the magnetic field and were prevented
from being eluted are freed in some manner such that they can be
eluted and recovered. In certain embodiments, the non-target cells
are labelled and depleted from the heterogeneous population of
cells.
[0540] In certain embodiments, the isolation or separation is
carried out using a system, device, or apparatus that carries out
one or more of the isolation, cell preparation, separation,
processing, incubation, culture, and/or formulation steps of the
methods. In some aspects, the system is used to carry out each of
these steps in a closed or sterile environment, for example, to
minimize error, user handling and/or contamination. In one example,
the system is a system as described in WO2009/072003 or US
20110003380.
[0541] In some embodiments, the system or apparatus carries out one
or more, e.g., all, of the isolation, processing, engineering, and
formulation steps in an integrated or self-contained system, and/or
in an automated or programmable fashion. In some aspects, the
system or apparatus includes a computer and/or computer program in
communication with the system or apparatus, which allows a user to
program, control, assess the outcome of, and/or adjust various
aspects of the processing, isolation, engineering, and formulation
steps.
[0542] In some aspects, the separation and/or other steps is
carried out using CliniMACS.RTM. system (Miltenyi Biotec), for
example, for automated separation of cells on a clinical-scale
level in a closed and sterile system. Components can include an
integrated microcomputer, magnetic separation unit, peristaltic
pump, and various pinch valves. The integrated computer in some
aspects controls all components of the instrument and directs the
system to perform repeated procedures in a standardized sequence.
The magnetic separation unit in some aspects includes a movable
permanent magnet and a holder for the selection column. The
peristaltic pump controls the flow rate throughout the tubing set
and, together with the pinch valves, ensures the controlled flow of
buffer through the system and continual suspension of cells.
[0543] The CliniMACS.RTM. system in some aspects uses
antibody-coupled magnetizable particles that are supplied in a
sterile, non-pyrogenic solution. In some embodiments, after
labelling of cells with magnetic particles the cells are washed to
remove excess particles. A cell preparation bag is then connected
to the tubing set, which in turn is connected to a bag containing
buffer and a cell collection bag. The tubing set consists of
pre-assembled sterile tubing, including a pre-column and a
separation column, and are for single use only. After initiation of
the separation program, the system automatically applies the cell
sample onto the separation column. Labelled cells are retained
within the column, while unlabeled cells are removed by a series of
washing steps. In some embodiments, the cell populations for use
with the methods described herein are unlabeled and are not
retained in the column. In some embodiments, the cell populations
for use with the methods described herein are labeled and are
retained in the column. In some embodiments, the cell populations
for use with the methods described herein are eluted from the
column after removal of the magnetic field, and are collected
within the cell collection bag.
[0544] In certain embodiments, separation and/or other steps are
carried out using the CliniMACS Prodigy.RTM. system (Miltenyi
Biotec). The CliniMACS Prodigy.RTM. system in some aspects is
equipped with a cell processing unity that permits automated
washing and fractionation of cells by centrifugation. The CliniMACS
Prodigy.RTM. system can also include an onboard camera and image
recognition software that determines the optimal cell fractionation
endpoint by discerning the macroscopic layers of the source cell
product. For example, peripheral blood may be automatically
separated into erythrocytes, white blood cells and plasma layers.
The CliniMACS Prodigy.RTM. system can also include an integrated
cell cultivation chamber which accomplishes cell culture protocols
such as, e.g., cell differentiation and expansion, antigen loading,
and long-term cell culture. Input ports can allow for the sterile
removal and replenishment of media and cells can be monitored using
an integrated microscope (see, e.g., Klebanoff et al. (2012) J
Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82,
and Wang et al. (2012) J Immunother. 35(9):689-701).
[0545] In some embodiments, a cell population described herein is
collected and enriched (or depleted) via flow cytometry, in which
cells stained for multiple cell surface markers are carried in a
fluidic stream. In some embodiments, a cell population described
herein is collected and enriched (or depleted) via preparative
scale (FACS)-sorting. In certain embodiments, a cell population
described herein is collected and enriched (or depleted) by use of
microelectromechanical systems (MEMS) chips in combination with a
FACS-based detection system (see, e.g., WO 2010/033140, Cho et al.
(2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton.
1(5):355-376. In both cases, cells can be labeled with multiple
markers, allowing for the isolation of well-defined T cell subsets
at high purity.
[0546] In some embodiments, the antibodies or binding partners are
labeled with one or more detectable marker, to facilitate
separation for positive and/or negative selection. For example,
separation may be based on binding to fluorescently labeled
antibodies. In some examples, separation of cells based on binding
of antibodies or other binding partners specific for one or more
cell surface markers are carried in a fluidic stream, such as by
fluorescence-activated cell sorting (FACS), including preparative
scale (FACS) and/or microelectromechanical systems (MEMS) chips,
e.g., in combination with a flow-cytometric detection system. Such
methods allow for positive and negative selection based on multiple
markers simultaneously.
[0547] In some embodiments, the preparation methods include steps
for freezing, e.g., cryopreserving, the cells, either before or
after isolation, incubation, and/or engineering. In some
embodiments, the freeze and subsequent thaw step removes
granulocytes and, to some extent, monocytes in the cell population.
In some embodiments, the cells are suspended in a freezing
solution, e.g., following a washing step to remove plasma and
platelets. Any of a variety of known freezing solutions and
parameters in some aspects may be used. One example involves using
PBS containing 20% DMSO and 8% human serum albumin (HSA), or other
suitable cell freezing media. This is then diluted 1:1 with media
so that the final concentration of DMSO and HSA are 10% and 4%,
respectively. The cells are then frozen to -80.degree. C. at a rate
of 1.degree. C. per minute and stored in the vapor phase of a
liquid nitrogen storage tank.
[0548] In some embodiments, the provided methods include
cultivation, incubation, culture, and/or genetic engineering steps.
For example, in some embodiments, provided are methods for
incubating and/or engineering the depleted cell populations and
culture-initiating compositions.
[0549] Thus, in some embodiments, the cell populations are
incubated in a culture-initiating composition. The incubation
and/or engineering may be carried out in a culture vessel, such as
a unit, chamber, well, column, tube, tubing set, valve, vial,
culture dish, bag, or other container for culture or cultivating
cells.
[0550] In some embodiments, the cells are incubated and/or cultured
prior to or in connection with genetic engineering. The incubation
steps can include culture, cultivation, stimulation, activation,
and/or propagation. In some embodiments, the compositions or cells
are incubated in the presence of stimulating conditions or a
stimulatory agent. Such conditions include those designed to induce
proliferation, expansion, activation, and/or survival of cells in
the population, to mimic antigen exposure, and/or to prime the
cells for genetic engineering, such as for the introduction of a
recombinant antigen receptor.
[0551] The conditions can include one or more of particular media,
temperature, oxygen content, carbon dioxide content, time, agents,
e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory
factors, such as cytokines, chemokines, antigens, binding partners,
fusion proteins, recombinant soluble receptors, and any other
agents designed to activate the cells.
[0552] In some embodiments, the stimulating conditions or agents
include one or more agent, e.g., ligand, which is capable of
stimulating or activating an intracellular signaling domain of a
TCR complex. In some aspects, the agent turns on or initiates
TCR/CD3 intracellular signaling cascade in a T cell. Such agents
can include antibodies, such as those specific for a TCR component
and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for
example, bound to solid support such as a bead, and/or one or more
cytokines. Optionally, the expansion method may further comprise
the step of adding anti-CD3 and/or anti-CD28 antibody to the
culture medium (e.g., at a concentration of at least about 0.5
ng/mL). In some embodiments, the stimulating agents include IL-2
and/or IL-15, for example, an IL-2 concentration of at least about
10 units/mL.
[0553] In some aspects, incubation is carried out in accordance
with techniques such as those described in U.S. Pat. No. 6,040,177
to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9):
651-660, Terakura et al. (2012) Blood.1:72-82, and/or Wang et al.
(2012) J Immunother. 35(9):689-701.
[0554] In some embodiments, the T cells are expanded by adding to
the culture-initiating composition feeder cells, such as
non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such
that the resulting population of cells contains at least about 5,
10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in
the initial population to be expanded); and incubating the culture
(e.g. for a time sufficient to expand the numbers of T cells). In
some aspects, the non-dividing feeder cells can comprise
gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC
are irradiated with gamma rays in the range of about 3000 to 3600
rads to prevent cell division. In some aspects, the feeder cells
are added to culture medium prior to the addition of the
populations of T cells.
[0555] In some embodiments, the stimulating conditions include
temperature suitable for the growth of human T lymphocytes, for
example, at least about 25 degrees Celsius, generally at least
about 30 degrees, and generally at or about 37 degrees Celsius.
Optionally, the incubation may further comprise adding non-dividing
EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can
be irradiated with gamma rays in the range of about 6000 to 10,000
rads. The LCL feeder cells in some aspects is provided in any
suitable amount, such as a ratio of LCL feeder cells to initial T
lymphocytes of at least about 10:1.
[0556] In embodiments, antigen-specific T cells, such as
antigen-specific CD4+ and/or CD8+ T cells, are obtained by
stimulating naive or antigen specific T lymphocytes with antigen.
For example, antigen-specific T cell lines or clones can be
generated to cytomegalovirus antigens by isolating T cells from
infected subjects and stimulating the cells in vitro with the same
antigen.
[0557] C. Engineered Cells, Vectors and Compositions for
Multi-Targeting
[0558] Also provided are cells such as engineered cells that can
bind to and/or target multiple antigens. In some embodiments,
improved selectivity and specificity is achieved through strategies
targeting multiple antigens. Such strategies generally involve
multiple antigen-binding domains, which typically are present on
distinct genetically engineered antigen receptors and specifically
bind to distinct antigens. In some embodiments, the cells are
engineered with the ability to bind more than one antigen. For
example, in some embodiments, the cells are engineered to express
multispecific binding molecules. In some embodiments, the cells
express multiple binding molecules, e.g., recombinant receptors,
each of which can target one antigen or multiple antigens, e.g.,
one receptor that targets ROR1, such as any described herein, and
another receptor that targets another antigen, e.g., tumor antigen.
In some aspects, a plurality of genetically engineered antigen
receptors are introduced into the cell, which specifically bind to
different antigens, each expressed in or on the disease or
condition to be targeted with the cells or tissues or cells
thereof. Such features can in some aspects address or reduce the
likelihood of off-target effects or increase efficacy. For example,
where a single antigen expressed in a disease or condition is also
expressed on or in non-diseased or normal cells, such
multi-targeting approaches can provide selectivity for desired cell
types by requiring binding via multiple antigen receptors in order
to activate the cell or induce a particular effector function. In
some embodiments, a plurality of cells can be engineered to express
one or more different binding molecules, e.g., recombinant
receptors, each of which can target one antigen or multiple
antigens.
[0559] Also provided are multispecific cells containing any of the
binding molecules described herein, such as cells containing a cell
surface protein including the anti-ROR1 antibody or an
antigen-binding fragment thereof and an additional cell surface
protein, such as an additional chimeric receptor, which binds to a
different antigen or a different epitope on ROR1. In some
embodiments, provided are compositions of cells that express
recombinant receptors, wherein one or more of the binding
molecules, multispecific binding molecules and/or recombinant
receptors bind and/or target ROR1. Also provided are compositions
of cells containing a plurality of cells that express one or more
different binding molecules, e.g., recombinant receptors that can
target one or multiple antigens. In some embodiments, the
multispecific binding molecules and/or recombinant receptors target
one or more different epitopes on ROR1.
[0560] In some embodiments, provided are composition of cells,
wherein each type of cell expresses one or more binding molecules,
e.g., recombinant receptors. In some embodiments, the cell
comprises (e.g., has been transformed with) one or more vectors
comprising one or more nucleic acid that encodes one or more an
amino acid sequence comprising one or more antibodies and/or
portions thereof, e.g., antigen-binding fragments thereof. In some
embodiments, one or more such cells are provided. In some
embodiments, a composition containing one or more such cells is
provided. In some embodiments, the one or more cells can express
different antibodies, or the same antibody. In some embodiments,
each of the cells expresses one or more antibodies, such as more
than one antibody. In some embodiments, each of the cells expresses
a multispecific binding molecule, e.g., a multispecific receptor,
e.g., CAR.
[0561] In some embodiments, the cells include multi-targeting
strategies that target ROR1 and a second or additional antigen
associated with a particular disease or condition. In some
embodiments, the second or additional antigen is targeted by a
multispecific binding molecule and/or multiple binding molecules
and/or a plurality of cells, e.g., one or more cells, each
engineered to express one or more recombinant receptors. In some
embodiments, a recombinant receptor targeting a second or
additional antigen is expressed on the same cell as a ROR1 binding
molecule, or on a different cell.
[0562] In some embodiments, among the second or additional antigens
for multi-targeting strategies includes those in which at least one
of the antigens is a universal tumor antigen, or a family member
thereof. In some embodiments, the second or additional antigen is
an antigen expressed on a tumor. In some embodiments, the
ROR1-binding molecules provided herein target an antigen on the
same tumor type as the second or additional antigen. In some
embodiments, the second or additional antigen may also be a
universal tumor antigen or may be a tumor antigen specific to a
tumor type. In some embodiments, the cell further comprises an
additional genetically engineered antigen receptor that recognizes
a second or additional antigen expressed on a disease or condition
to be treated and induces a stimulatory or activating signal.
[0563] Exemplary antigens include CD4, CD5, CD8, CD14, CD15, CD19,
CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46,
CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC-1, Ia, HM1.24,
HLA-DR, tenascin, an angiogenesis factor, VEGF, PIGF, ED-B
fibronectin, an oncogene, an oncogene product, CD66a-d, necrosis
antigens, Ii, IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4), TRAIL-R2
(DR5), B cell maturation antigen (BCMA), Her2, L1-CAM, mesothelin,
CEA, hepatitis B surface antigen, anti-folate receptor, CD24, CD30,
CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, erbB dimers,
EGFR vIII, FBP, FCRL5, FCRH5, fetal acetylcholine receptor, GD2,
GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain,
Lewis Y, L1-cell adhesion molecule (L1-CAM), Melanoma-associated
antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed
antigen of melanoma (PRAME), survivin, EGP2, EGP40, TAG72, B7-H6,
IL-13 receptor a2 (IL-13Ra2), CA9, CD171, G250/CAIX, HLA-AI MAGE
A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8,
avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2D
ligands, dual antigen, an antigen associated with a universal tag,
a cancer-testes antigen, MUC1, MUC16, NY-ESO-1, MART-1, gp100,
oncofetal antigen, VEGF-R2, carcinoembryonic antigen (CEA),
prostate specific antigen, PSMA, Her2/neu, estrogen receptor,
progesterone receptor, ephrinB2, CD123, c-Met, GD-2, 0-acetylated
GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1,
hTERT, MDM2, CYP1B, WT1, livin, AFP, p53, cyclin (Dl), CS-1, BCMA,
BAFF-R, TACI, CD56, TIM-3, CD123, L1-cell adhesion molecule,
MAGE-A1, MAGE A3, a cyclin, such as cyclin A1 (CCNA1) and/or a
pathogen-specific antigen, biotinylated molecules, molecules
expressed by HIV, HCV, HBV and/or other pathogens, and/or in some
aspects, neoepitopes or neoantigens thereof. In some embodiments,
the antigen is associated with or is a universal tag.
[0564] In some embodiments, the plurality of antigens, e.g., the
first antigen, e.g., ROR1, and the second or additional antigens,
are expressed on the cell, tissue, or disease or condition being
targeted, such as on the cancer cell. In some aspects, the cell,
tissue, disease or condition is or is associated with a cancer or a
tumor. One or more of the plurality of antigens generally also is
expressed on a cell which it is not desired to target with the cell
therapy, such as a normal or non-diseased cell or tissue, and/or
the engineered cells themselves. In such embodiments, by requiring
ligation of multiple receptors to achieve a response of the cell,
specificity and/or efficacy is/are achieved.
[0565] In some embodiments, the cells and methods include
multi-targeting strategies, such as expression of two or more
genetically engineered receptors on the cell, each recognizing a
different antigen and typically each including a different
intracellular signaling component. Such multi-targeting strategies
are described, for example, in WO 2014055668 (describing
combinations of activating and costimulatory CARs, e.g., targeting
two different antigens present individually on off-target, e.g.,
normal cells, but present together only on cells of the disease or
condition to be treated) and Fedorov et al., Sci. Transl. Medicine,
5(215) (December, 2013) (describing cells expressing an activating
and an inhibitory CAR, such as those in which the activating CAR
binds to one antigen expressed on both normal or non-diseased cells
and cells of the disease or condition to be treated, and the
inhibitory CAR binds to another antigen expressed only on the
normal cells or cells which it is not desired to treat).
[0566] In some embodiments, a plurality of cells, each engineered
to express one or more recombinant receptors, are provided. For
example, in some embodiments, one cell is engineered to express a
binding molecule that binds and/or targets ROR1, and another cell
is engineered to express a binding molecule that binds and/or
targets an additional or second antigen. In some embodiments, the
cells can each express a multispecific binding molecule, e.g., a
multispecific recombinant receptor, where one or more of the target
antigen is ROR1. In some of such embodiments, the plurality of
cells can be administered together or separately. In some
embodiments, some of the plurality of cells are administered
simultaneously or concurrently with other cells, e.g., administered
on the same day, and/or sequentially with or intermittently with,
in any order, another engineered cell in the plurality. For
example, in some embodiments, an engineered cell expressing a
ROR1-binding molecule, e.g., CAR, is administered simultaneously
with or sequentially with, in any order, another engineered cell
expressing a binding molecule that binds a different target antigen
or a different epitope on ROR1. In some embodiments, the plurality
of cells can be in the same composition or in different
compositions. Exemplary compositions of the cells include
compositions described in Section III below.
III. PHARMACEUTICAL COMPOSITIONS
[0567] Also provided are compositions including the ROR1-binding
molecules, immunoconjugates, recombinant receptors, and engineered
cells, including pharmaceutical compositions and formulations. Also
provided are compositions comprising engineered cells that express
the ROR1-binding molecules provided herein, such as recombinant
receptors (e.g., CARs), including pharmaceutical compositions and
formulations.
[0568] Provided are pharmaceutical formulations comprising a
ROR1-binding molecule (e.g., antibody), an immunoconjugate, a
recombinant receptor (e.g., chimeric antigen receptor), engineered
cells expressing said molecules (e.g., antibody or recombinant
receptor), a plurality of engineered cells expressing said
molecules (e.g., recombinant receptor) and/or additional agents for
combination treatment or therapy. The pharmaceutical compositions
and formulations generally include one or more optional
pharmaceutically acceptable carrier or excipient. In some
embodiments, the composition includes at least one additional
therapeutic agent.
[0569] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0570] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0571] In some aspects, the choice of carrier is determined in part
by the particular cell, binding molecule, and/or antibody, and/or
by the method of administration. Accordingly, there are a variety
of suitable formulations. For example, the pharmaceutical
composition can contain preservatives. Suitable preservatives may
include, for example, methylparaben, propylparaben, sodium
benzoate, and benzalkonium chloride. In some aspects, a mixture of
two or more preservatives is used. The preservative or mixtures
thereof are typically present in an amount of about 0.0001% to
about 2% by weight of the total composition. Carriers are
described, e.g., by Remington's Pharmaceutical Sciences 16th
edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers
are generally nontoxic to recipients at the dosages and
concentrations employed, and include, but are not limited to:
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride; benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
polyethylene glycol (PEG).
[0572] Buffering agents in some aspects are included in the
compositions. Suitable buffering agents include, for example,
citric acid, sodium citrate, phosphoric acid, potassium phosphate,
and various other acids and salts. In some aspects, a mixture of
two or more buffering agents is used. The buffering agent or
mixtures thereof are typically present in an amount of about 0.001%
to about 4% by weight of the total composition. Methods for
preparing administrable pharmaceutical compositions are known.
Exemplary methods are described in more detail in, for example,
Remington: The Science and Practice of Pharmacy, Lippincott
Williams & Wilkins; 21st ed. (May 1, 2005).
[0573] Formulations of the antibodies described herein can include
lyophilized formulations and aqueous solutions.
[0574] The formulation or composition may also contain more than
one active ingredient useful for the particular indication,
disease, or condition being treated with the binding molecules or
cells, preferably those with activities complementary to the
binding molecule or cell, where the respective activities do not
adversely affect one another. Such active ingredients are suitably
present in combination in amounts that are effective for the
purpose intended. Thus, in some embodiments, the pharmaceutical
composition further includes other pharmaceutically active agents
or drugs, such as chemotherapeutic agents, e.g., asparaginase,
busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin,
fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel,
rituximab, vinblastine, vincristine, etc. In some embodiments, the
cells or antibodies are administered in the form of a salt, e.g., a
pharmaceutically acceptable salt. Suitable pharmaceutically
acceptable acid addition salts include those derived from mineral
acids, such as hydrochloric, hydrobromic, phosphoric,
metaphosphoric, nitric, and sulphuric acids, and organic acids,
such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic,
glycolic, gluconic, succinic, and arylsulphonic acids, for example,
p-toluenesulphonic acid.
[0575] Active ingredients may be entrapped in microcapsules, in
colloidal drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions. In certain embodiments, the pharmaceutical
composition is formulated as an inclusion complex, such as
cyclodextrin inclusion complex, or as a liposome. Liposomes can
serve to target the host cells (e.g., T-cells or NK cells) to a
particular tissue. Many methods are available for preparing
liposomes, such as those described in, for example, Szoka et al.,
Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Pat. Nos.
4,235,871, 4,501,728, 4,837,028, and 5,019,369.
[0576] The pharmaceutical composition in some aspects can employ
time-released, delayed release, and sustained release delivery
systems such that the delivery of the composition occurs prior to,
and with sufficient time to cause, sensitization of the site to be
treated. Many types of release delivery systems are available and
known. Such systems can avoid repeated administrations of the
composition, thereby increasing convenience to the subject and the
physician.
[0577] The pharmaceutical composition in some embodiments contains
the binding molecules and/or cells in amounts effective to treat or
prevent the disease or condition, such as a therapeutically
effective or prophylactically effective amount. Therapeutic or
prophylactic efficacy in some embodiments is monitored by periodic
assessment of treated subjects. For repeated administrations over
several days or longer, depending on the condition, the treatment
is repeated until a desired suppression of disease symptoms occurs.
However, other dosage regimens may be useful and can be determined.
The desired dosage can be delivered by a single bolus
administration of the composition, by multiple bolus
administrations of the composition, or by continuous infusion
administration of the composition.
[0578] In certain embodiments, in the context of genetically
engineered cells containing the binding molecules, a subject is
administered the range of at or about one million to at or about
100 billion cells, such as, e.g., at or about 1 million to at or
about 50 billion cells (e.g., at or about 5 million cells, at or
about 25 million cells, at or about 500 million cells, at or about
1 billion cells, at or about 5 billion cells, at or about 20
billion cells, at or about 30 billion cells, at or about 40 billion
cells, or a range defined by any two of the foregoing values), such
as at or about 10 million to at or about 100 billion cells (e.g.,
at or about 20 million cells, at or about 30 million cells, at or
about 40 million cells, at or about 60 million cells, at or about
70 million cells, at or about 80 million cells, at or about 90
million cells, at or about 10 billion cells, at or about 25 billion
cells, at or about 50 billion cells, at or about 75 billion cells,
at or about 90 billion cells, or a range defined by any two of the
foregoing values), and in some cases at or about 100 million cells
to at or about 50 billion cells (e.g., at or about 120 million
cells, at or about 250 million cells, at or about 350 million
cells, at or about 450 million cells, at or about 650 million
cells, at or about 800 million cells, at or about 900 million
cells, at or about 3 billion cells, at or about 30 billion cells,
at or about 45 billion cells) or any value in between these ranges,
and/or such a number of cells per kilogram of body weight of the
subject.
[0579] The may be administered using standard administration
techniques, formulations, and/or devices. Provided are formulations
and devices, such as syringes and vials, for storage and
administration of the compositions. Administration of the cells can
be autologous or heterologous. For example, immune cells,
immunoresponsive cells or progenitors can be obtained from one
subject, and administered to the same subject or a different,
compatible subject. Peripheral blood derived immunoresponsive cells
or their progeny (e.g., in vivo, ex vivo or in vitro derived) can
be administered via localized injection, including catheter
administration, systemic injection, localized injection,
intravenous injection, or parenteral administration. When
administering a therapeutic composition (e.g., a pharmaceutical
composition containing a genetically modified immune cell such as a
T cell), it will generally be formulated in a unit dosage
injectable form (solution, suspension, emulsion).
[0580] Formulations include those for oral, intravenous,
intraperitoneal, subcutaneous, pulmonary, transdermal,
intramuscular, intranasal, buccal, sublingual, or suppository
administration. In some embodiments, the cell populations are
administered parenterally. The term "parenteral," as used herein,
includes intravenous, intramuscular, subcutaneous, rectal, vaginal,
intracranial, intrathoracic, and intraperitoneal administration. In
some embodiments, the cell populations are administered to a
subject using peripheral systemic delivery by intravenous,
intraperitoneal, or subcutaneous injection.
[0581] Compositions in some embodiments are provided as sterile
liquid preparations, e.g., isotonic aqueous solutions, suspensions,
emulsions, dispersions, or viscous compositions, which may in some
aspects be buffered to a selected pH. Liquid preparations are
normally easier to prepare than gels, other viscous compositions,
and solid compositions. Additionally, liquid compositions are
somewhat more convenient to administer, especially by injection.
Viscous compositions, on the other hand, can be formulated within
the appropriate viscosity range to provide longer contact periods
with specific tissues. Liquid or viscous compositions can comprise
carriers, which can be a solvent or dispersing medium containing,
for example, water, saline, phosphate buffered saline, polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycol)
and suitable mixtures thereof.
[0582] Sterile injectable solutions can be prepared by
incorporating the binding molecule in a solvent, such as in
admixture with a suitable carrier, diluent, or excipient such as
sterile water, physiological saline, glucose, dextrose, or the
like. The compositions can also be lyophilized. The compositions
can contain auxiliary substances such as wetting, dispersing, or
emulsifying agents (e.g., methylcellulose), pH buffering agents,
gelling or viscosity enhancing additives, preservatives, flavoring
agents, colors, and the like, depending upon the route of
administration and the preparation desired. Standard texts may in
some aspects be consulted to prepare suitable preparations.
[0583] Various additives which enhance the stability and sterility
of the compositions, including antimicrobial preservatives,
antioxidants, chelating agents, and buffers, can be added.
Prevention of the action of microorganisms can be ensured by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, and the like. Prolonged
absorption of the injectable pharmaceutical form can be brought
about by the use of agents delaying absorption, for example,
aluminum monostearate and gelatin.
[0584] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules.
[0585] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0586] Also provided are pharmaceutical compositions for
combination therapy. Any of the additional agents for combination
therapy described herein, such as agents described in Section IV.B,
can be prepared and administered as one or more pharmaceutical
compositions, with the ROR1-binding molecule (e.g., antibody),
immunoconjugate, recombinant receptor (e.g., chimeric antigen
receptor) and/or engineered cells expressing said molecules (e.g.,
recombinant receptor) described herein. The combination therapy can
be administered in one or more pharmaceutical compositions, e.g.,
where the binding molecules, recombinant receptors and/or cells are
in the same pharmaceutical composition as the additional agent, or
in separate pharmaceutical compositions. For example, in some
embodiments, the additional agent is an additional engineered cell,
e.g., cell engineered to express a different recombinant receptor
that targets a different antigen or a different epitope on ROR1,
and is administered in the same composition or in a separate
composition. In some embodiments, each of the pharmaceutical
composition is formulated in a suitable formulation according to
the particular binding molecule, recombinant receptor, cell, e.g.,
engineered cell, and/or additional agent, and the particular dosage
regimen and/or method of delivery.
IV. METHODS AND USES
[0587] Also provided methods, such as methods of treatment, of
using and uses of the ROR1-binding molecules, immunoconjugates,
recombinant receptors, engineered cells, and pharmaceutical
compositions and formulations thereof, such as in the treatment of
diseases, conditions, and disorders in which ROR1 is expressed,
and/or detection, diagnostic, and prognostic methods. Also provided
are methods of combination therapy and/or treatment.
[0588] A. Therapeutic and Prophylactic Methods and Uses
[0589] Also provided are methods of administering and uses, such as
therapeutic and prophylactic uses, of the ROR1-binding molecules,
including the anti-ROR1 antibodies, e.g., antibody fragments and
proteins containing the same such as the recombinant receptors
(e.g., CARs), engineered cells expressing the recombinant receptors
(e.g., CARs), plurality of engineered cells expressing the
receptors, and/or compositions comprising the same. Such methods
and uses include therapeutic methods and uses, for example,
involving administration of the molecules (e.g., ROR1-binding
molecules, recombinant receptors), cells (e.g., engineered cells),
or compositions containing the same, to a subject having a disease,
condition, or disorder associated with ROR1 such as a disease,
condition, or disorder associated with ROR1 expression, and/or in
which cells or tissues express, e.g., specifically express ROR1. In
some embodiments, the molecule, cell, and/or composition is
administered in an effective amount to effect treatment of the
disease or disorder. Provided herein are uses of the binding
molecules (e.g., anti-ROR1 antibodies or antigen-binding fragments
thereof), recombinant receptors (e.g., CARs), and cells (e.g.,
engineered cells) in such methods and treatments, and in the
preparation of a medicament in order to carry out such therapeutic
methods. Uses include uses of the binding molecules, CARs,
antibodies, and cells in such methods and treatments, and in the
preparation of a medicament in order to carry out such therapeutic
methods. In some embodiments, the methods are carried out by
administering the binding molecules or cells, or compositions
comprising the same, to the subject having, having had, or
suspected of having the disease or condition. In some embodiments,
the methods thereby treat the disease or condition or disorder in
the subject. Also provided herein are of use of any of the
compositions, such as pharmaceutical compositions provided herein,
for the treatment of a disease or disorder associated with ROR1,
for example, a ROR1-expressing cancer.
[0590] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to complete or
partial amelioration or reduction of a disease or condition or
disorder, or a symptom, adverse effect or outcome, or phenotype
associated therewith. Desirable effects of treatment include, but
are not limited to, preventing occurrence or recurrence of disease,
alleviation of symptoms, diminishment of any direct or indirect
pathological consequences of the disease, preventing metastasis,
decreasing the rate of disease progression, amelioration or
palliation of the disease state, and remission or improved
prognosis. The terms do not imply complete curing of a disease or
complete elimination of any symptom or effect(s) on all symptoms or
outcomes.
[0591] As used herein, "delaying development of a disease" means to
defer, hinder, slow, retard, stabilize, suppress and/or postpone
development of the disease (such as cancer). This delay can be of
varying lengths of time, depending on the history of the disease
and/or individual being treated. As is evident to one skilled in
the art, a sufficient or significant delay can, in effect,
encompass prevention, in that the individual does not develop the
disease. For example, a late stage cancer, such as development of
metastasis, may be delayed.
[0592] "Preventing," as used herein, includes providing prophylaxis
with respect to the occurrence or recurrence of a disease in a
subject that may be predisposed to the disease but has not yet been
diagnosed with the disease. In some embodiments, the provided
molecules and compositions are used to delay development of a
disease or to slow the progression of a disease.
[0593] As used herein, to "suppress" a function or activity is to
reduce the function or activity when compared to otherwise same
conditions except for a condition or parameter of interest, or
alternatively, as compared to another condition. For example, an
antibody or composition or cell which suppresses tumor growth
reduces the rate of growth of the tumor compared to the rate of
growth of the tumor in the absence of the antibody or composition
or cell.
[0594] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, binding molecule, antibody, or cells, or composition,
in the context of administration, refers to an amount effective, at
dosages/amounts and for periods of time necessary, to achieve a
desired result, such as a therapeutic or prophylactic result.
[0595] A "therapeutically effective amount" of an agent, e.g., a
pharmaceutical formulation, antibody, or cells, refers to an amount
effective, at dosages and for periods of time necessary, to achieve
a desired therapeutic result, such as for treatment of a disease,
condition, or disorder, and/or pharmacokinetic or pharmacodynamic
effect of the treatment. The therapeutically effective amount may
vary according to factors such as the disease state, age, sex, and
weight of the subject, and the populations of cells administered.
In some embodiments, the provided methods involve administering the
molecules, cells, and/or compositions at effective amounts, e.g.,
therapeutically effective amounts.
[0596] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically but not necessarily,
since a prophylactic dose is used in subjects prior to or at an
earlier stage of disease, the prophylactically effective amount
will be less than the therapeutically effective amount.
[0597] As used herein, a "subject" is a mammal, such as a human or
other animal, and typically is human.
[0598] Among the diseases to be treated are any ROR1-associated
disease or condition or disease or condition in which ROR1 is
specifically expressed. In certain diseases and conditions, ROR1 is
expressed on malignant cells and cancers. In some embodiments, the
disease or condition is a ROR1-expressing cancer. Among the
ROR1-associated diseases or conditions that can be treated include,
but are not limited to, B cell leukemia, lymphoma, B cell chronic
lymphocytic leukemia (CLL), acute myeloid leukemia (AML), acute
lymphocytic leukemia (ALL), Burkitt's Lymphoma, mantle cell
lymphoma (MCL), non-small cell lung cancer (NSCLC), neuroblastoma,
renal cell carcinoma, colon cancer, colorectal cancer, breast
cancer, epithelial squamous cell cancer, melanoma, myeloma, stomach
cancer, brain cancer, lung cancer, pancreatic cancer, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, prostate
cancer, testicular cancer, thyroid cancer, uterine cancer, adrenal
cancer and head and neck cancer. In some embodiments, the disease
or condition to be treated is selected from a CLL, a MCL, an
ovarian cancer, a lung cancer, a pancreatic cancer or a breast
cancer. In some embodiments, the disease or condition to be treated
is a CLL. In some embodiments, the disease or condition to be
treated is a MCL. In some embodiments, the disease or condition to
be treated is an ovarian cancer. In some embodiments, the disease
or condition to be treated is a lung cancer. In some embodiments,
the disease or condition to be treated is a pancreatic cancer. In
some embodiments, the disease or condition to be treated is a
breast cancer. In some embodiments, the disease or condition to be
treated is a lung cancer, and the lung cancer is selected from
among lung adenocarcinoma, adenocarcinoma, squamous cell carcinoma,
small cell carcinoma, non-small cell lung cancer (NSCLC) and
atypical carcinoid. In some embodiments, the disease or condition
to be treated is a lung cancer, and the lung cancer is anon-small
cell lung cancer (NSCLC). In some embodiments, the disease or
condition to be treated is a breast cancer, and the breast cancer
is a triple-negative breast cancer.
[0599] In some aspects, in certain diseases and conditions, ROR1 is
expressed on malignant cells and cancers, including cancers
associated with a solid tumor or a hematologic malignancy. In some
embodiments, the disease or condition is a ROR1-expressing cancer.
In some embodiments among the disease or disorder to be treated is
a cancer, such as a ROR1-expressing cancer. In some embodiments,
the cancer is associated with a solid tumor or a hematologic
malignancy.
[0600] In some embodiments, the cancer is associated with a
ROR1-expressing solid tumor. In some embodiments, the disease or
disorder to be treated is a cancer associated with a solid tumor,
such as neuroblastoma, renal cell carcinoma, colon cancer,
colorectal cancer, breast cancer, epithelial squamous cell cancer,
melanoma, myeloma, stomach cancer, brain cancer, lung cancer,
pancreatic cancer, cervical cancer, ovarian cancer, liver cancer,
bladder cancer, prostate cancer, testicular cancer, thyroid cancer,
uterine cancer, adrenal cancer and head and neck cancer. In some
embodiments, the lung cancer is a non-small cell lung cancer
(NSCLC). In some embodiments, the breast cancer is a triple
negative breast cancer (TNBC). In some aspects, triple negative
breast cancer refers to a breast cancer that is negative for
expression of estrogen receptors, progesterone receptors and human
epidermal growth factor receptor 2 (HER2) overexpression.
[0601] In some embodiments, the cancer is associated with a
ROR1-expressing hematologic malignancy. In some embodiments, the
disease or disorder associated with ROR1 is a B cell-related
disorder. In some embodiments, the disease or disorder to be
treated is a cancer associated with a hematologic malignancy, such
as B cell leukemia, lymphoma, B cell chronic lymphocytic leukemia
(CLL), acute myeloid leukemia (AML), acute lymphocytic leukemia
(ALL), Burkitt's Lymphoma or mantle cell lymphoma (MCL).
[0602] In some embodiments, the methods include administering
engineered cells (e.g. T cells) expressing a provided binding
molecule to a subject having a ROR1-associated disease or
condition, such as a ROR1-expressing cancer. In particular
embodiments, the engineered cells (e.g. T cells) express a
recombinant receptor, such as a CAR, containing as an
antigen-binding domain a provided antibody or antigen-binding
fragment thereof (e.g. scFv). In some embodiments, the engineered
cells are autologous to the subject being treated. In some
embodiments, the engineered cells are allogeneic to the subject
being treated, in which case the cells are obtained from the
diseased subject and engineered with the recombinant receptor (e.g.
CAR) prior to administration of the engineered cells.
[0603] In some contexts, engineering primary T cells obtained from
subjects with diseases or disorders such as chronic lymphocytic
leukemia (CLL) to express recombinant receptors has been reported
to result in engineered T cells with impaired function, compared to
primary T cells from subjects with other hematologic malignancies
(see, e.g., Gorgun et al., J Clin Invest. 2005 July;
115(7):1797-805; Ramsay et al., J Clin Invest. 2008 July;
118(7):2427-37; Riches et al., Discov Med. 2013 December;
16(90):295-302). In some cases, as described herein, the provided
recombinant receptors can be expressed from primary T cells
obtained from subjects with CLL, without observed impairment in
function. In some aspects, engineered cells expressing provided
binding molecules, e.g., recombinant receptors targeting ROR1, can
be successfully generated using cells from subjects with diseases
or disorders such as CLL.
[0604] In some embodiments, the methods may identify a subject who
has, is suspected to have, or is at risk for developing a
ROR1-associated disease or disorder. Hence, provided are methods
for identifying subjects with diseases or disorders associated with
elevated ROR1 expression and selecting them for treatment with a
provided ROR1 binding molecule, including any of the anti-ROR1
antibodies, e.g., antibody fragments and proteins containing the
same such as the chimeric receptors, and/or engineered cells
expressing the recombinant receptors.
[0605] For example, a subject may be screened for the presence of a
disease or disorder associated with elevated ROR1 expression, such
as a ROR1-expressing cancer. In some embodiments, the methods
include screening for or detecting the presence of a
ROR1-associated disease, e.g. a tumor. Thus, in some aspects, a
sample may be obtained from a patient suspected of having a disease
or disorder associated with elevated ROR1 expression and assayed
for the expression level of ROR1. In some aspects, a subject who
tests positive for a ROR1-associated disease or disorder may be
selected for treatment by the present methods, and may be
administered a therapeutically effective amount of an anti-ROR1
antibody, a ROR1-targeting CAR, cells containing a CAR or a
pharmaceutical composition thereof as described herein. In some
embodiments, the methods can be used to monitor the size or density
of a ROR1-expressing tissue, e.g. tumor, over time, e.g., before,
during, or after treatment by the methods.
[0606] In some embodiments, the subject has persistent or relapsed
disease, e.g., following treatment with another ROR1-specific
antibody and/or cells expressing a ROR1-targeting chimeric receptor
and/or other therapy, including chemotherapy, radiation, and/or
hematopoietic stem cell transplantation (HSCT), e.g., allogenic
HSCT. In some embodiments, the administration effectively treats
the subject despite the subject having become resistant to another
ROR1-targeted therapy. In some embodiments, the subject has not
relapsed but is determined to be at risk for relapse, such as at a
high risk of relapse, and thus the compound or composition is
administered prophylactically, e.g., to reduce the likelihood of or
prevent relapse.
[0607] In some embodiments, the treatment does not induce an immune
response by the subject to the therapy, and/or does not induce such
a response to a degree that prevents effective treatment of the
disease or condition. In some aspects, the degree of immunogenicity
and/or graft versus host response is less than that observed with a
different but comparable treatment. For example, in the case of
adoptive cell therapy using cells expressing CARs including the
provided anti-ROR1 antibodies, the degree of immunogenicity in some
embodiments is reduced compared to CARs including a different
antibody that binds to a similar, e.g., overlapping epitope and/or
that competes for binding to ROR1 with the provided antibody, such
as a mouse or rabbit or humanized antibody.
[0608] In some embodiments, the methods include adoptive cell
therapy, whereby genetically engineered cells expressing the
provided ROR1-targeting receptors (e.g., containing ROR1-targeting
antibody or fragment thereof) are administered to subjects. Such
administration can promote activation of the cells (e.g., T cell
activation) in a ROR1-targeting manner, such that the cells of the
disease or disorder are targeted for destruction.
[0609] Thus, the provided methods and uses include methods and uses
for adoptive cell therapy. In some embodiments, the methods include
administration of the cells or a composition containing the cells
to a subject, tissue, or cell, such as one having, at risk for, or
suspected of having the disease, condition or disorder. In some
embodiments, the cells, populations, and compositions are
administered to a subject having the particular disease or
condition to be treated, e.g., via adoptive cell therapy, such as
adoptive T cell therapy. In some embodiments, the cells or
compositions are administered to the subject, such as a subject
having or at risk for the disease or condition. In some aspects,
the methods thereby treat, e.g., ameliorate one or more symptom of
the disease or condition, such as by lessening tumor burden in a
ROR1-expressing cancer.
[0610] Methods for administration of cells for adoptive cell
therapy are known and may be used in connection with the provided
methods and compositions. For example, adoptive T cell therapy
methods are described, e.g., in US Patent Application Publication
No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to
Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See,
e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933;
Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9;
Davila et al. (2013) PLoS ONE 8(4): e61338.
[0611] In some embodiments, the cell therapy, e.g., adoptive cell
therapy, e.g., adoptive T cell therapy, is carried out by
autologous transfer, in which the cells are isolated and/or
otherwise prepared from the subject who is to receive the cell
therapy, or from a sample derived from such a subject. Thus, in
some aspects, the cells are derived from a subject, e.g., patient,
in need of a treatment and the cells, following isolation and
processing are administered to the same subject.
[0612] In some embodiments, the cell therapy, e.g., adoptive cell
therapy, e.g., adoptive T cell therapy, is carried out by
allogeneic transfer, in which the cells are isolated and/or
otherwise prepared from a subject other than a subject who is to
receive or who ultimately receives the cell therapy, e.g., a first
subject. In such embodiments, the cells then are administered to a
different subject, e.g., a second subject, of the same species. In
some embodiments, the first and second subjects are genetically
identical. In some embodiments, the first and second subjects are
genetically similar. In some embodiments, the second subject
expresses the same HLA class or supertype as the first subject.
[0613] In some embodiments, the subject, to whom the cells, cell
populations, or compositions are administered is a primate, such as
a human. In some embodiments, the primate is a monkey or an ape.
The subject can be male or female and can be any suitable age,
including infant, juvenile, adolescent, adult, and geriatric
subjects. In some embodiments, the subject is a non-primate mammal,
such as a rodent. In some examples, the patient or subject is a
validated animal model for disease, adoptive cell therapy, and/or
for assessing toxic outcomes such as cytokine release syndrome
(CRS).
[0614] The ROR1-binding molecules, such as antibodies and chimeric
receptors containing the antibodies and cells expressing the same,
can be administered by any suitable means, for example, by
injection, e.g., intravenous or subcutaneous injections,
intraocular injection, periocular injection, subretinal injection,
intravitreal injection, trans-septal injection, subscleral
injection, intrachoroidal injection, intracameral injection,
subconjectval injection, subconjuntival injection, sub-Tenon's
injection, retrobulbar injection, peribulbar injection, or
posterior juxtascleral delivery. In some embodiments, they are
administered by parenteral, intrapulmonary, and intranasal, and, if
desired for local treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, intracranial, intrathoracic, or
subcutaneous administration. Dosing and administration may depend
in part on whether the administration is brief or chronic. Various
dosing schedules include but are not limited to single or multiple
administrations over various time-points, bolus administration, and
pulse infusion.
[0615] For the prevention or treatment of disease, the appropriate
dosage of the binding molecule or cell may depend on the type of
disease to be treated, the type of binding molecule, the severity
and course of the disease, whether the binding molecule is
administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the binding
molecule, and the discretion of the attending physician. The
compositions and molecules and cells are in some embodiments
suitably administered to the patient at one time or over a series
of treatments.
[0616] Depending on the type and severity of the disease, dosages
of antibodies may include at or about 1 .mu.g/kg to at or about 15
mg/kg (e.g. at or about 0.1 mg/kg at or about 10 mg/kg), at or
about 1 .mu.g/kg to at or about 100 mg/kg or more, at or about 0.05
mg/kg to at or about 10 mg/kg, at or about 0.5 mg/kg, at or about
2.0 mg/kg, at or about 4.0 mg/kg or at or about 10 mg/kg. Multiple
doses may be administered intermittently, e.g. every week or every
three weeks. An initial higher loading dose, followed by one or
more lower doses may be administered.
[0617] In certain embodiments, the cells, or individual populations
of sub-types of cells, are administered to the subject at a range
of at or about 0.1 million to at or about 100 billion cells and/or
that amount of cells per kilogram of body weight of the subject,
such as, e.g., at or about 0.1 million to at or about 50 billion
cells (e.g., at or about 5 million cells, at or about 25 million
cells, at or about 500 million cells, at or about 1 billion cells,
at or about 5 billion cells, at or about 20 billion cells, at or
about 30 billion cells, at or about 40 billion cells, or a range
defined by any two of the foregoing values), at or about 1 million
to at or about 50 billion cells (e.g., at or about 5 million cells,
at or about 25 million cells, at or about 500 million cells, at or
about 1 billion cells, at or about 5 billion cells, at or about 20
billion cells, at or about 30 billion cells, at or about 40 billion
cells, or a range defined by any two of the foregoing values), such
as at or about 10 million to at or about 100 billion cells (e.g.,
at or about 20 million cells, at or about 30 million cells, at or
about 40 million cells, at or about 60 million cells, at or about
70 million cells, at or about 80 million cells, at or about 90
million cells, at or about 10 billion cells, at or about 25 billion
cells, at or about 50 billion cells, at or about 75 billion cells,
at or about 90 billion cells, or a range defined by any two of the
foregoing values), and in some cases at or about 100 million cells
to at or about 50 billion cells (e.g., at or about 120 million
cells, at or about 250 million cells, at or about 350 million
cells, at or about 650 million cells, at or about 800 million
cells, at or about 900 million cells, at or about 3 billion cells,
at or about 30 billion cells, at or about 45 billion cells) or any
value in between these ranges and/or per kilogram of body weight of
the subject. Dosages may vary depending on attributes particular to
the disease or disorder and/or patient and/or other treatments. In
some embodiments, such values refer to numbers of recombinant
receptor-expressing cells; in other embodiments, they refer to
number of T cells or PBMCs or total cells administered.
[0618] In some embodiments, for example, where the subject is a
human, the dose includes fewer than about 5.times.10.sup.8 total
recombinant receptor (e.g., CAR)-expressing cells, T cells, or
peripheral blood mononuclear cells (PBMCs), e.g., in the range of
at or about 1.times.10.sup.6 to at or about 5.times.10.sup.8 such
cells, such as at or about 2.times.10.sup.6, 5.times.10.sup.6,
1.times.10.sup.7, 5.times.10.sup.7, 1.times.10.sup.8,
1.5.times.10.sup.8, or 5.times.10.sup.8 total such cells, or the
range between any two of the foregoing values. In some embodiments,
for example, where the subject is a human, the dose includes more
than at or about 1.times.10.sup.6 total recombinant receptor (e.g.,
CAR)-expressing cells, T cells, or peripheral blood mononuclear
cells (PBMCs) and fewer than at or about 2.times.10.sup.9 total
recombinant receptor (e.g., CAR)-expressing cells, T cells, or
peripheral blood mononuclear cells (PBMCs), e.g., in the range of
at or about 2.5.times.10.sup.7 to at or about 1.2.times.10.sup.9
such cells, such as at or about 2.5.times.10.sup.7,
5.times.10.sup.7, 1.times.10.sup.8, 1.5.times.10.sup.8,
8.times.10.sup.8, or 1.2.times.10.sup.9 total such cells, or the
range between any two of the foregoing values.
[0619] In some embodiments, the dose of genetically engineered
cells comprises from at least or at least about or is
1.times.10.sup.5 to at or about 5.times.10.sup.8 total
CAR-expressing (CAR.sup.+) T cells, from at or about
1.times.10.sup.5 to at or about 2.5.times.10.sup.8 total CAR.sup.+
T cells, from at or about 1.times.10.sup.5 to at or about
1.times.10.sup.8 total CAR.sup.+ T cells, from at or about
1.times.10.sup.5 to at or about 5.times.10.sup.7 total CAR.sup.+ T
cells, from at or about 1.times.10.sup.5 to at or about
2.5.times.10.sup.7 total CAR.sup.+ T cells, from at or about
1.times.10.sup.5 to at or about 1.times.10.sup.7 total CAR.sup.+ T
cells, from at or about 1.times.10.sup.5 to at or about
5.times.10.sup.6 total CAR.sup.+ T cells, from at or about
1.times.10.sup.5 to at or about 2.5.times.10.sup.6 total CAR.sup.+
T cells, from at or about 1.times.10.sup.5 to at or about
1.times.10.sup.6 total CAR.sup.+ T cells, from at or about
1.times.10.sup.6 to at or about 5.times.10.sup.8 total CAR.sup.+ T
cells, from at or about 1.times.10.sup.6 to at or about
2.5.times.10.sup.8 total CAR.sup.+ T cells, from at or about
1.times.10.sup.6 to at or about 1.times.10.sup.8 total CAR.sup.+ T
cells, from at or about 1.times.10.sup.6 to at or about
5.times.10.sup.7 total CAR.sup.+ T cells, from at or about
1.times.10.sup.6 to at or about 2.5.times.10.sup.7 total CAR+ T
cells, from at or about 1.times.10.sup.6 to at or about
1.times.10.sup.7 total CAR.sup.+ T cells, from at or about
1.times.10.sup.6 to at or about 5.times.10.sup.6 total CAR.sup.+ T
cells, from at or about 1.times.10.sup.6 to at or about
2.5.times.10.sup.6 total CAR.sup.+ T cells, from at or about
2.5.times.10.sup.6 to at or about 5.times.10.sup.8 total CAR.sup.+
T cells, from at or about 2.5.times.10.sup.6 to at or about
2.5.times.10.sup.8 total CAR.sup.+ T cells, from at or about
2.5.times.10.sup.6 to at or about 1.times.10.sup.8 total CAR.sup.+
T cells, from at or about 2.5.times.10.sup.6 to at or about
5.times.10.sup.7 total CAR.sup.+ T cells, from at or about
2.5.times.10.sup.6 to at or about 2.5.times.10.sup.7 total
CAR.sup.+ T cells, from at or about 2.5.times.10.sup.6 to at or
about 1.times.10.sup.7 total CAR.sup.+ T cells, from at or about
2.5.times.10.sup.6 to at or about 5.times.10.sup.6 total CAR.sup.+
T cells, from at or about 5.times.10.sup.6 to at or about
5.times.10.sup.8 total CAR.sup.+ T cells, from at or about
5.times.10.sup.6 to at or about 2.5.times.10.sup.8 total CAR.sup.+
T cells, from at or about 5.times.10.sup.6 to at or about
1.times.10.sup.8 total CAR.sup.+ T cells, from at or about
5.times.10.sup.6 to at or about 5.times.10.sup.7 total CAR.sup.+ T
cells, from at or about 5.times.10.sup.6 to at or about
2.5.times.10.sup.7 total CAR.sup.+ T cells, from at or about
5.times.10.sup.6 to at or about 1.times.10.sup.7 total CAR.sup.+ T
cells, from at or about 1.times.10.sup.7 to at or about
5.times.10.sup.8 total CAR.sup.+ T cells, from at or about
1.times.10.sup.7 to at or about 2.5.times.10.sup.8 total CAR.sup.+
T cells, from at or about 1.times.10.sup.7 to at or about
1.times.10.sup.8 total CAR.sup.+ T cells, from at or about
1.times.10.sup.7 to at or about 5.times.10.sup.7 total CAR.sup.+ T
cells, from at or about 1.times.10.sup.7 to at or about
2.5.times.10.sup.7 total CAR.sup.+ T cells, from at or about
2.5.times.10.sup.7 to at or about 5.times.10.sup.7 total CAR.sup.+
T cells, from at or about 2.5.times.10.sup.7 to at or about
2.5.times.10.sup.8 total CAR.sup.+ T cells, from at or about
2.5.times.10.sup.7 to at or about 1.times.10.sup.8 total CAR.sup.+
T cells, from at or about 2.5.times.10.sup.7 to at or about
5.times.10.sup.7 total CAR.sup.+ T cells, from at or about
5.times.10.sup.7 to at or about 5.times.10.sup.7 total CAR.sup.+ T
cells, from at or about 5.times.10.sup.7 to at or about
2.5.times.10.sup.8 total CAR.sup.+ T cells, from at or about
5.times.10.sup.7 to at or about 1.times.10.sup.8 total CAR.sup.+ T
cells, from at or about 1.times.10.sup.8 to at or about
5.times.10.sup.7 total CAR.sup.+ T cells, from at or about
1.times.10.sup.8 to at or about 2.5.times.10.sup.8 total CAR.sup.+
T cells, from at or about or 2.5.times.10.sup.8 to at or about
5.times.10.sup.7 total CAR.sup.+ T cells. In some embodiments, the
dose of genetically engineered cells comprises from or from about
2.5.times.10.sup.7 to at or about 1.5.times.10.sup.8 total
CAR.sup.+ T cells, such as from or from about 5.times.10.sup.7 to
or to about 1.times.10.sup.8 total CAR.sup.+ T cells.
[0620] In some embodiments, the dose of genetically engineered
cells comprises at least at or about 1.times.10.sup.5 CAR.sup.+
cells, at least at or about 2.5.times.10.sup.5 CAR.sup.+ cells, at
least at or about 5.times.10.sup.5 CAR.sup.+ cells, at least at or
about 1.times.10.sup.6 CAR.sup.+ cells, at least at or about
2.5.times.10.sup.6 CAR.sup.+ cells, at least at or about
5.times.10.sup.6 CAR.sup.+ cells, at least at or about
1.times.10.sup.7 CAR.sup.+ cells, at least at or about
2.5.times.10.sup.7 CAR.sup.+ cells, at least at or about
5.times.10.sup.7 CAR.sup.+ cells, at least at or about
1.times.10.sup.8 CAR.sup.+ cells, at least at or about
1.5.times.10.sup.8 CAR.sup.+ cells, at least at or about
2.5.times.10.sup.8 CAR.sup.+ cells, or at least at or about
5.times.10.sup.8 CAR.sup.+ cells.
[0621] In some embodiments, the cell therapy comprises
administration of a dose comprising a number of cell from or from
about 1.times.10.sup.5 to or to about 5.times.10.sup.7 total
recombinant receptor-expressing cells, total T cells, or total
peripheral blood mononuclear cells (PBMCs), from or from about
5.times.10.sup.5 to or to about 1.times.10.sup.7 total recombinant
receptor-expressing cells, total T cells, or total peripheral blood
mononuclear cells (PBMCs) or from or from about 1.times.10.sup.6 to
or to about 1.times.10.sup.7 total recombinant receptor-expressing
cells, total T cells, or total peripheral blood mononuclear cells
(PBMCs), each inclusive. In some embodiments, the cell therapy
comprises administration of a dose of cells comprising a number of
cells at least or at least about 1.times.10.sup.5 total recombinant
receptor-expressing cells, total T cells, or total peripheral blood
mononuclear cells (PBMCs), such at least or at least
1.times.10.sup.6, at least or at least about 1.times.10.sup.7, at
least or at least about 1.times.10.sup.8 of such cells. In some
embodiments, the number is with reference to the total number of
CD3.sup.+ or CD8.sup.+, in some cases also recombinant
receptor-expressing (e.g. CAR.sup.+) cells. In some embodiments,
the cell therapy comprises administration of a dose comprising a
number of cell from or from about 1.times.10.sup.5 to or to about
5.times.10.sup.8 CD3.sup.+ or CD8.sup.+ total T cells or CD3.sup.+
or CD8.sup.+ recombinant receptor-expressing cells, from or from
about 5.times.10.sup.5 to or to about 1.times.10.sup.7 CD3.sup.+ or
CD8.sup.+ total T cells or CD3.sup.+ or CD8.sup.+ recombinant
receptor-expressing cells, or from or from about 1.times.10.sup.6
to or to about 1.times.10.sup.7 CD3.sup.+ or CD8.sup.+ total T
cells or CD3.sup.+ or CD8.sup.+ recombinant receptor-expressing
cells, each inclusive. In some embodiments, the cell therapy
comprises administration of a dose comprising a number of cell from
or from about 1.times.10.sup.5 to or to about 5.times.10.sup.8
total CD3.sup.+/CAR.sup.+ or CD8.sup.+/CAR.sup.+ cells, from or
from about 5.times.10.sup.5 to or to about 1.times.10.sup.7 total
CD3.sup.+/CAR.sup.+ or CD8.sup.+/CAR.sup.+ cells, or from or from
about 1.times.10.sup.6 to or to about 1.times.10.sup.7 total
CD3.sup.+/CAR.sup.+ or CD8.sup.+/CAR.sup.+ cells, each
inclusive.
[0622] In some embodiments, the T cells of the dose include CD4+ T
cells, CD8+ T cells or CD4+ and CD8+ T cells.
[0623] In some embodiments, for example, where the subject is
human, the CD8.sup.+ T cells of the dose, including in a dose
including CD4.sup.+ and CD8.sup.+ T cells, includes between at or
about 1.times.10.sup.6 and at or about 5.times.10.sup.8 total
recombinant receptor (e.g., CAR)-expressing CD8.sup.+ cells, e.g.,
in the range of from at or about 5.times.10.sup.6 to at or about
1.times.10.sup.8 such cells, such as 1.times.10.sup.7,
2.5.times.10.sup.7, 5.times.10.sup.7, 7.5.times.10.sup.7,
1.times.10.sup.8, 1.5.times.10.sup.8, or 5.times.10.sup.8 total
such cells, or the range between any two of the foregoing values.
In some embodiments, the patient is administered multiple doses,
and each of the doses or the total dose can be within any of the
foregoing values. In some embodiments, the dose of cells comprises
the administration of from or from about 1.times.10.sup.7 to or to
about 0.75.times.10.sup.8 total recombinant receptor-expressing
CD8.sup.+ T cells, from or from about 1.times.10.sup.7 to or to
about 5.times.10.sup.7 total recombinant receptor-expressing
CD8.sup.+ T cells, from or from about 1.times.10.sup.7 to or to
about 0.25.times.10.sup.8 total recombinant receptor-expressing
CD8.sup.+ T cells, each inclusive. In some embodiments, the dose of
cells comprises the administration of at or about 1.times.10.sup.7,
2.5.times.10.sup.7, 5.times.10.sup.7, 7.5.times.10.sup.7,
1.times.10.sup.8, 1.5.times.10.sup.8, 2.5.times.10.sup.8, or
5.times.10.sup.8 total recombinant receptor-expressing CD8.sup.+ T
cells.
[0624] In some embodiments, the dose of cells, e.g., recombinant
receptor-expressing T cells, is administered to the subject as a
single dose or is administered only one time within a period of two
weeks, one month, three months, six months, 1 year or more.
[0625] In some embodiments, the cells or antibodies are
administered as part of a combination treatment, such as
simultaneously with or sequentially with, in any order, another
therapeutic intervention, such as another antibody or engineered
cell or receptor or agent, such as a cytotoxic or therapeutic
agent.
[0626] The cells or antibodies in some embodiments are
co-administered with one or more additional therapeutic agents or
in connection with another therapeutic intervention, either
simultaneously or sequentially in any order. In some contexts, the
cells are co-administered with another therapy sufficiently close
in time such that the cell populations enhance the effect of one or
more additional therapeutic agents, or vice versa. In some
embodiments, the cells or antibodies are administered prior to the
one or more additional therapeutic agents. In some embodiments, the
cells or antibodies are administered after to the one or more
additional therapeutic agents.
[0627] Once the cells are administered to a mammal (e.g., a human),
the biological activity of the engineered cell populations and/or
antibodies in some aspects is measured by any of a number of known
methods. Parameters to assess include specific binding of an
engineered or natural T cell or other immune cell to antigen, in
vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow
cytometry. In certain embodiments, the ability of the engineered
cells to destroy target cells can be measured using any suitable
known methods, such as cytotoxicity assays described in, for
example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702
(2009), and Herman et al. J. Immunological Methods, 285(1): 25-40
(2004). In certain embodiments, the biological activity of the
cells also can be measured by assaying expression and/or secretion
of certain cytokines, such as CD 107a, IFN.gamma., IL-2, and TNF.
In some aspects the biological activity is measured by assessing
clinical outcome, such as reduction in tumor burden or load.
[0628] In certain embodiments, engineered cells are modified in any
number of ways, such that their therapeutic or prophylactic
efficacy is increased. For example, the engineered CAR or TCR
expressed by the population in some embodiments are conjugated
either directly or indirectly through a linker to a targeting
moiety. The practice of conjugating compounds, e.g., the CAR or
TCR, to targeting moieties is known. See, for instance, Wadwa et
al., J. Drug Targeting 3: 1 1 1 (1995), and U.S. Pat. No.
5,087,616.
[0629] B. Combination Therapy
[0630] Also provided are methods of combination therapy that
includes administering and uses, such as therapeutic and
prophylactic uses, of the ROR1-binding molecules, including the
anti-ROR1 antibodies, e.g., antibody fragments, conjugates, and
proteins containing the same, such as the recombinant receptors
(e.g., CARs), engineered cells expressing the recombinant receptors
(e.g., CARs), plurality of engineered cells expressing the
receptors, and/or compositions comprising the same.
[0631] In some embodiments, the ROR1-binding molecule (e.g.,
antibody), immunoconjugate, recombinant receptor (e.g., chimeric
antigen receptor) and/or engineered cells expressing said molecules
(e.g., recombinant receptor) described herein are administered as
part of a combination treatment or combination therapy, such as
simultaneously with, sequentially with or intermittently with, in
any order, one or more additional therapeutic intervention. In some
embodiments, the one or more additional therapeutic intervention
includes, for example, an antibody, an engineered cell, a receptor
and/or an agent, such as a cell expressing a recombinant receptor,
and/or cytotoxic or therapeutic agent, e.g., a chemotherapeutic
agent. In some embodiments, the combination therapy includes
administration of one or more additional agents, therapies and/or
treatments, e.g., any of the additional agents, therapy and/or
treatments described herein. In some embodiments, the combination
therapy includes administration of one or more additional agents
for treatment or therapy, such as an immunomodulatory agent, immune
checkpoint inhibitor, adenosine pathway or adenosine receptor
antagonist or agonist and kinase inhibitors. In some embodiments,
the combination treatment or combination therapy includes an
additional treatment, such as a surgical treatment, transplant,
and/or radiation therapy. Also provided are methods of combination
treatment or combination therapy that includes administering the
binding molecules (e.g., ROR1-binding molecules), recombinant
receptors, cells and/or compositions described herein and one or
more additional therapeutic interventions.
[0632] In some embodiments, the additional agent for combination
treatment or combination therapy enhances, boosts and/or promotes
the efficacy and/or safety of the therapeutic effect of binding
molecules, recombinant receptors, cells and/or compositions. In
some embodiments, the additional agent enhances or improves the
efficacy, survival or persistence of the administered cells, e.g.,
cells expressing the binding molecule or a recombinant receptor. In
some embodiments, the additional agent is selected from among a
protein phosphatase inhibitor, a kinase inhibitor, a cytokine, an
immunomodulator, or an agent that decreases the level or activity
of a regulatory T (Treg) cell. In some embodiments, the additional
agent enhances safety, by virtue of reducing or ameliorating
adverse effects of the administered binding molecules, recombinant
receptors, cells and/or compositions. In some embodiments, the
additional agent can treat the same disease, condition or a
comorbidity. In some embodiments, the additional agent can
ameliorate, reduce or eliminate one or more toxicities, adverse
effects or side effects that are associated with administration of
the binding molecules, recombinant receptors, cells and/or
compositions, e.g., CAR-expressing cells.
[0633] In some embodiments, the additional therapy, treatment or
agent includes chemotherapy, radiation therapy, surgery,
transplantation, adoptive cell therapy, antibodies, cytotoxic
agents, chemotherapeutic agents, cytokines, growth inhibitory
agents, anti-hormonal agents, kinase inhibitors, anti-angiogenic
agents, cardioprotectants, immunostimulatory agents,
immunosuppressive agents, immune checkpoint inhibitors,
antibiotics, angiogenesis inhibitors, metabolic modulators or other
therapeutic agents or any combination thereof. In some embodiments,
the additional agent is a protein, a peptide, a nucleic acid, a
small molecule agent, a cell, a toxin, a lipid, a carbohydrate or
combinations thereof, or any other type of therapeutic agent, e.g.
radiation. In some embodiments, the additional therapy, agent or
treatment includes surgery, chemotherapy, radiation therapy,
transplantation, administration of cells expressing a recombinant
receptor, e.g., CAR, kinase inhibitor, immune checkpoint inhibitor,
mTOR pathway inhibitor, immunosuppressive agents, immunomodulators,
antibodies, immunoablative agents, antibodies and/or antigen
binding fragments thereof, antibody conjugates, other antibody
therapies, cytotoxins, steroids, cytokines, peptide vaccines,
hormone therapy, antimetabolites, metabolic modulators, drugs that
inhibit either the calcium dependent phosphatase calcineurin or the
p70S6 kinase FK506) or inhibit the p70S6 kinase, alkylating agents,
anthracyclines, vinca alkaloids, proteasome inhibitors, GITR
agonists, protein tyrosine phosphatase inhibitors, protein kinase
inhibitors, an oncolytic virus, and/or other types of
immunotherapy. In some embodiments, the additional agent or
treatment is bone marrow transplantation, T cell ablative therapy
using chemotherapy agents such as, fludarabine, external-beam
radiation therapy (XRT), cyclophosphamide, and/or antibody
therapy.
[0634] In some embodiments, the cells, binding molecules (e.g.,
ROR1-binding molecules), recombinant receptors and/or compositions,
e.g., CAR-expressing cells, are administered in combination with
other engineered cells, e.g., other CAR-expressing cells. In some
embodiments, the additional agent is a kinase inhibitor, e.g., an
inhibitor of Bruton's tyrosine kinase (Btk), e.g., ibrutinib. In
some embodiments, the additional agent is an adenosine pathway or
adenosine receptor antagonist or agonist. In some embodiments, the
additional agent is an immunomodulator such as thalidomide or a
thalidomide derivative (e.g., lenalidomide). In some embodiments,
the additional therapy, agent or treatment is a cytotoxic or
chemotherapy agent, a biologic therapy (e.g., antibody, e.g.,
monoclonal antibody, or cellular therapy), or an inhibitor (e.g.,
kinase inhibitor).
[0635] In some embodiments, a chemotherapeutic agent (sometimes
referred to as a cytotoxic agent) is administered to the subject to
disrupt a lesion. In certain embodiments, the lesion is tumor. In
particular embodiments, the lesion is cancerous. In particular
embodiments, the chemotherapeutic agent is any agent known to those
of skill in the art to be effective for the treatment, prevention
or amelioration of hyperproliferative disorders such as cancer.
Chemotherapeutic agents include, but are not limited to, small
molecules, synthetic drugs, peptides, polypeptides, proteins,
nucleic acids (e.g., DNA and RNA polynucleotides including, but not
limited to, antisense nucleotide sequences, triple helices and
nucleotide sequences encoding biologically active proteins,
polypeptides or peptides), antibodies, synthetic or natural
inorganic molecules, mimetic agents, and synthetic or natural
organic molecules. In particular embodiments, chemotherapeutic
drugs include alkylating agents, anthracyclines, cytoskeletal
disruptors (taxanes), epothilones, histone deacetylase inhibitors,
topoisomerase inhibitors, topoisomerase II inhibitors, kinase
inhibitors, nucleotide analogs and precursor analogs, peptide
antibiotics, platinum-based agents, and vinca alkaloids and
derivatives.
[0636] In certain embodiments, a lesion is disrupted by
administering a chemotherapeutic agent to modulate genetically
engineered cells in vivo. Chemotherapeutic agents may include, but
are not limited to, abarelix, aldesleukin, alemtuzumab,
alitretinoin, allopurinol, altretamine, amifostine, anastrozole,
arsenic trioxide, asparaginase, BCG live, bevaceizumab, bexarotene,
bleomycin, bortezomib, busulfan, calusterone, camptothecin,
capecitabine, carboplatin, carmustine, celecoxib, cetuximab,
chlorambucil, cinacalcet, cisplatin, cladribine, cyclophosphamide,
cytarabine, dacarbazine, dactinomycin, darbepoetin alfa,
daunorubicin, denileukin diftitox, dexrazoxane, docetaxel,
doxorubicin, dromostanolone, Elliott's B solution, epirubicin,
epoetin alfa, estramustine, etoposide, exemestane, filgrastim,
floxuridine, fludarabine, fluorouracil, fulvestrant, gemcitabine,
gemtuzumab ozogamicin, gefitinib, goserelin, hydroxyurea,
ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib, interferon
alfa-2a, interferon alfa-2b, irinotecan, letrozole, leucovorin,
levamisole, lomustine, meclorethamine, megestrol, melphalan,
mercaptopurine, mesna, methotrexate, methoxsalen,
methylprednisolone, mitomycin C, mitotane, mitoxantrone,
nandrolone, nofetumomab, oblimersen, oprelvekin, oxaliplatin,
paclitaxel, pamidronate, pegademase, pegaspargase, pegfilgrastim,
pemetrexed, pentostatin, pipobroman, plicamycin, polifeprosan,
porfimer, procarbazine, quinacrine, rasburicase, rituximab,
sargramostim, streptozocin, talc, tamoxifen, tarceva, temozolomide,
teniposide, testolactone, thioguanine, thiotepa, topotecan,
toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard,
valrubicin, vinblastine, vincristine, vinorelbine, and
zoledronate.
[0637] In some embodiments, exemplary chemotherapeutic agents
include an anthracycline (e.g., doxorubicin, such as liposomal
doxorubicin); a vinca alkaloid (e.g., vinblastine, vincristine,
vindesine, vinorelbine); an alkylating agent (e.g.,
cyclophosphamide, decarbazine, melphalan, ifosfamide,
temozolomide); an immune cell antibody (e.g., alemtuzumab,
gemtuzumab, rituximab, tositumomab); an antimetabolite (including,
e.g., folic acid antagonists, pyrimidine analogs, purine analogs
and adenosine deaminase inhibitors such as fludarabine); a TNFR
glucocorticoid induced TNFR related protein (GITR) agonist; a
proteasome inhibitor (e.g., aclacinomycin A, gliotoxin or
bortezomib); an immunomodulatory such as thalidomide or a
thalidomide derivative (e.g., lenalidomide).
[0638] In some embodiments, the additional therapy or treatment is
cell therapy, e.g., adoptive cell therapy. In some embodiments, the
additional therapy includes administration of engineered cells,
e.g., additional CAR-expressing cell. In some embodiments, the
additional engineered cell is a CAR-expressing cell that expresses
the same or different recombinant receptor as the engineered cells
provided herein, e.g., anti-ROR1 CAR-expressing cells. In some
embodiments, the recombinant receptor, e.g., CAR, expressed on the
additional engineered cell, recognizes a different antigen and/or
epitope. In some embodiments, the recombinant receptor, e.g., CAR,
expressed on the additional engineered cell, recognizes a different
epitope of the same antigen as the recombinant receptors described
herein, e.g., ROR1. In some embodiments, the recombinant receptor,
e.g., CAR, expressed on the additional engineered cell, recognizes
a different antigen, e.g., a different tumor antigen or combination
of antigens. For example, in some embodiments, the recombinant
receptor, e.g., CAR, expressed on the additional engineered cell,
targets cancer cells that express early lineage markers, e.g.,
cancer stem cells, while other CAR-expressing cells target cancer
cells that express later lineage markers. In such embodiments, the
additional engineered cell is administered prior to, concurrently
with, or after administration (e.g., infusion) of the
CAR-expressing cells described herein. In some embodiments, the
additional engineered cell expresses allogeneic CAR.
[0639] In some embodiments, the configurations of one or more of
the CAR molecules comprise a primary intracellular signaling domain
and two or more, e.g., 2, 3, 4, or 5 or more, costimulatory
signaling domains. In some embodiments, the one or more of the CAR
molecules may have the same or a different primary intracellular
signaling domain, the same or different costimulatory signaling
domains, or the same number or a different number of costimulatory
signaling domains. In some embodiments, the one or more of the CAR
molecules can be configured as a split CAR, in which one of the CAR
molecules comprises an antigen binding domain and a costimulatory
domain (e.g., 4-1BB), while the other CAR molecule comprises an
antigen binding domain and a primary intracellular signaling domain
(e.g., CD3 zeta).
[0640] In some embodiments, the additional agent is any of the
multispecific binding molecules and/or cells engineered to express
one or more of the binding molecules described herein and/or cells
engineered to express additional binding molecules, e.g.,
recombinant receptors, e.g., CAR, that target a different antigen.
In some embodiments, the additional agent includes any of the cells
or plurality of cells described herein, e.g., in Section I.C. In
some embodiments, the additional agent is a cell engineered to
express a recombinant receptor, e.g., CAR, targeting a different
epitope and/or antigen, e.g., a different antigen associated with a
disease or condition. In some embodiments, the additional agent is
a cell engineered to express a recombinant receptor, e.g., CAR,
targeting a second or additional antigen expressed in the disease
or disorder, e.g., a cancer or a tumor.
[0641] In some embodiments, the additional agent is an
immunomodulatory agent. In some embodiments, the combination
therapy includes an immunomodulatory agent that can stimulate,
amplify and/or otherwise enhance an anti-tumor immune response,
e.g., anti-tumor immune response from the administered engineered
cells, such as by inhibiting immunosuppressive signaling or
enhancing immunostimulant signaling. In some embodiments, the
immunomodulatory agent is a peptide, protein or is a small
molecule. In some embodiments, the protein can be a fusion protein
or a recombinant protein. In some embodiments, the immunomodulatory
agent binds to an immunologic target, such as a cell surface
receptor expressed on immune cells, such a T cells, B cells or
antigen-presenting cells. For example, in some embodiments, the
immunomodulatory agent is an antibody or antigen-binding antibody
fragment, a fusion protein, a small molecule or a polypeptide. In
some embodiments, the binding molecules, recombinant receptors,
cells and/or compositions are administered in combination with an
additional agent that is an antibody or an antigen-binding fragment
thereof, such as a monoclonal antibody.
[0642] In some embodiments, the immunomodulatory agent blocks,
inhibits or counteracts a component of the immune checkpoint
pathway. The immune system has multiple inhibitory pathways that
are involved in maintaining self-tolerance and for modulating
immune responses. Tumors can use certain immune-checkpoint pathways
as a major mechanism of immune resistance, particularly against T
cells that are specific for tumor antigens (Pardoll (2012) Nature
Reviews Cancer 12:252-264), e.g., engineered cells such as
CAR-expressing cells. Because many such immune checkpoints are
initiated by ligand-receptor interactions, they can be readily
blocked by antibodies against the ligands and/or their
receptors.
[0643] Therefore, therapy with antagonistic molecules blocking an
immune checkpoint pathway, such as small molecules, nucleic acid
inhibitors (e.g., RNAi) or antibody molecules, are becoming
promising avenues of immunotherapy for cancer and other diseases.
In contrast to the majority of anti-cancer agents, checkpoint
inhibitors do not necessarily target tumor cells directly, but
rather target lymphocyte receptors or their ligands in order to
enhance the endogenous antitumor activity of the immune system.
[0644] As used herein, the term "immune checkpoint inhibitor"
refers to molecules that totally or partially reduce, inhibit,
interfere with or modulate one or more checkpoint proteins.
Checkpoint proteins regulate T-cell activation or function. These
proteins are responsible for co-stimulatory or inhibitory
interactions of T-cell responses Immune checkpoint proteins
regulate and maintain self-tolerance and the duration and amplitude
of physiological immune responses. In some embodiments, the subject
can be administered an additional agent that can enhance or boost
the immune response, e.g., immune response effected by the binding
molecules (e.g., ROR1-binding molecules), recombinant receptors,
cells and/or compositions provided herein, against a disease or
condition, e.g., a cancer, such as any described herein.
[0645] Immune checkpoint inhibitors include any agent that blocks
or inhibits in a statistically significant manner, the inhibitory
pathways of the immune system. Such inhibitors may include small
molecule inhibitors or may include antibodies, or antigen binding
fragments thereof, that bind to and block or inhibit immune
checkpoint receptors, ligands and/or receptor-ligand interaction.
In some embodiments, modulation, enhancement and/or stimulation of
particular receptors can overcome immune checkpoint pathway
components. Illustrative immune checkpoint molecules that may be
targeted for blocking, inhibition, modulation, enhancement and/or
stimulation include, but are not limited to, PD-1 (CD279), PD-L1
(CD274, B7-H1), PDL2 (CD273, B7-DC), CTLA-4, LAG-3 (CD223), TIM-3,
4-1BB (CD137), 4-1BBL (CD137L), GITR (TNFRSF18, AITR), CD40, OX40
(CD134, TNFRSF4), CXCR2, tumor associated antigens (TAA), B7-H3,
B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4, VISTA, KIR, 2B4 (belongs to
the CD2 family of molecules and is expressed on all NK,
.gamma..delta., and memory CD8+ (.alpha..beta.) T cells), CD160
(also referred to as BY55), CGEN-15049, CEACAM (e.g., CEACAM-1,
CEACAM-3 and/or CEACAM-5), TIGIT, LAIR1, CD160, 2B4, CD80, CD86,
B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR,
MHC class I, MHC class II, GAL9, adenosine, and a transforming
growth factor receptor (TGFR; e.g., TGFR beta) Immune checkpoint
inhibitors include antibodies, or antigen binding fragments
thereof, or other binding proteins that bind to and block or
inhibit and/or enhance or stimulate the activity of one or more of
any of the said molecules.
[0646] Exemplary immune checkpoint inhibitors include Tremelimumab
(CTLA-4 blocking antibody, also known as ticilimumab, CP-675,206),
anti-OX40, PD-L1 monoclonal antibody (Anti-B7-H1; MEDI4736),
MK-3475 (PD-1 blocker), nivolumab (anti-PD-1 antibody), CT-011
(anti-PD-1 antibody), BY55 monoclonal antibody, AMP224 (anti-PD-L1
antibody), BMS-936559 (anti-PD-L1 antibody), MPLDL3280A (anti-PD-L1
antibody), MSB0010718C (anti-PD-L1 antibody) and ipilimumab
(anti-CTLA-4 antibody, also known as Yervoy.RTM., MDX-010 and
MDX-101). Exemplary of immunomodulatory antibodies include, but are
not limited to, Daclizumab (Zenapax), Bevacizumab (Avastin
Basiliximab, Ipilimumab, Nivolumab, pembrolizumab, MPDL3280A,
Pidilizumab (CT-011), MK-3475, BMS-936559, MPDL3280A
(Atezolizumab), tremelimumab, IMP321, BMS-986016, LAG525, urelumab,
PF-05082566, TRX518, MK-4166, dacetuzumab (SGN-40), lucatumumab
(HCD122), SEA-CD40, CP-870, CP-893, MEDI6469, MEDI6383, MOXR0916,
AMP-224, MSB0010718C (Avelumab), MEDI4736, PDR001, rHIgMl2B7,
Ulocuplumab, BKT140, Varlilumab (CDX-1127), ARGX-110, MGA271,
lirilumab (BMS-986015, IPH2101), IPH2201, ARGX-115, Emactuzumab,
CC-90002 and MNRP1685A or an antibody-binding fragment thereof.
Other exemplary immunomodulators include, e.g., afutuzumab
(available from Roche.RTM.); pegfilgrastim (Neulasta.RTM.);
lenalidomide (CC-5013, Revlimid.RTM.); thalidomide (Thalomid.RTM.),
actimid (CC4047); and IRX-2 (mixture of human cytokines including
interleukin 1, interleukin 2, and interferon gamma, CAS
951209-71-5, available from IRX Therapeutics).
[0647] Programmed cell death 1 (PD-1) is an immune checkpoint
protein that is expressed in B cells, NK cells, and T cells
(Shinohara et al., 1995, Genomics 23:704-6; Blank et al., 2007,
Cancer Immunol Immunother 56:739-45; Finger et al., 1997, Gene
197:177-87; Pardoll (2012) Nature Reviews Cancer 12:252-264). The
major role of PD-1 is to limit the activity of T cells in
peripheral tissues during inflammation in response to infection, as
well as to limit autoimmunity. PD-1 expression is induced in
activated T cells and binding of PD-1 to one of its endogenous
ligands acts to inhibit T-cell activation by inhibiting stimulatory
kinases. PD-1 also acts to inhibit the TCR "stop signal". PD-1 is
highly expressed on Treg cells and may increase their proliferation
in the presence of ligand (Pardoll (2012) Nature Reviews Cancer
12:252-264). Anti-PD 1 antibodies have been used for treatment of
melanoma, non-small-cell lung cancer, bladder cancer, prostate
cancer, colorectal cancer, head and neck cancer, triple-negative
breast cancer, leukemia, lymphoma and renal cell cancer (Topalian
et al., 2012, N Engl J Med 366:2443-54; Lipson et al., 2013, Clin
Cancer Res 19:462-8; Berger et al., 2008, Clin Cancer Res
14:3044-51; Gildener-Leapman et al., 2013, Oral Oncol 49:1089-96;
Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85). Exemplary
anti-PD-1 antibodies include nivolumab (Opdivo by BMS),
pembrolizumab (Keytruda by Merck), pidilizumab (CT-011 by Cure
Tech), lambrolizumab (MK-3475 by Merck), and AMP-224 (Merck),
nivolumab (also referred to as Opdivo, BMS-936558 or MDX1106;
Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody
which specifically blocks PD-1. Nivolumab (clone 5C4) and other
human monoclonal antibodies that specifically bind to PD-1 are
described in U.S. Pat. No. 8,008,449 and WO2006/121168. Pidilizumab
(CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that
binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal
antibodies are described in WO2009/101611. Pembrolizumab (formerly
known as lambrolizumab, and also referred to as Keytruda, MK03475;
Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1.
Pembrolizumab and other humanized anti-PD-1 antibodies are
described in U.S. Pat. No. 8,354,509 and WO2009/114335. Other
anti-PD-1 antibodies include AMP 514 (Amplimmune), among others,
e.g., anti-PD-1 antibodies described in U.S. Pat. No. 8,609,089, US
2010028330, US 20120114649 and/or US 20150210769. AMP-224 (B7-DCIg;
Amplimmune; e.g., described in WO2010/027827 and WO2011/066342), is
a PD-L2 Fc fusion soluble receptor that blocks the interaction
between PD-1 and B7-H1.
[0648] PD-L1 (also known as CD274 and B7-H1) and PD-L2 (also known
as CD273 and B7-DC) are ligands for PD-1, found on activated T
cells, B cells, myeloid cells, macrophages, and some types of tumor
cells. Anti-tumor therapies have focused on anti-PD-L1 antibodies.
The complex of PD-1 and PD-L1 inhibits proliferation of CD8+ T
cells and reduces the immune response (Topalian et al., 2012, N
Engl J Med 366:2443-54; Brahmer et al., 2012, N Eng J Med
366:2455-65). Anti-PD-L1 antibodies have been used for treatment of
non-small cell lung cancer, melanoma, colorectal cancer, renal-cell
cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast
cancer, and hematologic malignancies (Brahmer et al., 2012, N Eng J
Med 366:2455-65; Ott et al., 2013, Clin Cancer Res 19:5300-9;
Radvanyi et al., 2013, Clin Cancer Res 19:5541; Menzies & Long,
2013, Ther Adv Med Oncol 5:278-85; Berger et al., 2008, Clin Cancer
Res 14:13044-51). Exemplary anti-PD-L1 antibodies include MDX-1105
(Medarex), MEDI4736 (Medimmune) MPDL3280A (Genentech), BMS-935559
(Bristol-Myers Squibb) and MSB0010718C. MEDI4736 (Medimmune) is a
human monoclonal antibody that binds to PD-L1, and inhibits
interaction of the ligand with PD-1. MDPL3280A (Genentech/Roche) is
a human Fc optimized IgG1 monoclonal antibody that binds to PD-L1.
MDPL3280A and other human monoclonal antibodies to PD-L1 are
described in U.S. Pat. No. 7,943,743 and U.S Publication No.
20120039906. Other anti-PD-L1 binding agents include YW243.55.570
(see WO2010/077634) and MDX-1105 (also referred to as BMS-936559,
and, e.g., anti-PD-L1 binding agents described in
WO2007/005874).
[0649] Cytotoxic T-lymphocyte-associated antigen (CTLA-4), also
known as CD152, is a co-inhibitory molecule that functions to
regulate T-cell activation. CTLA-4 is a member of the
immunoglobulin superfamily that is expressed exclusively on
T-cells. CTLA-4 acts to inhibit T-cell activation and is reported
to inhibit helper T-cell activity and enhance regulatory T-cell
immunosuppressive activity. Although the precise mechanism of
action of CTLA-4 remains under investigation, it has been suggested
that it inhibits T cell activation by outcompeting CD28 in binding
to CD80 and CD86, as well as actively delivering inhibitor signals
to the T cell (Pardoll (2012) Nature Reviews Cancer 12:252-264).
Anti-CTLA-4 antibodies have been used in clinical trials for the
treatment of melanoma, prostate cancer, small cell lung cancer,
non-small cell lung cancer (Robert & Ghiringhelli, 2009,
Oncologist 14:848-61; Ott et al., 2013, Clin Cancer Res 19:5300;
Weber, 2007, Oncologist 12:864-72; Wada et al., 2013, J Transl Med
11:89). A significant feature of anti-CTLA-4 is the kinetics of
anti-tumor effect, with a lag period of up to 6 months after
initial treatment required for physiologic response. In some cases,
tumors may actually increase in size after treatment initiation,
before a reduction is seen (Pardoll (2012) Nature Reviews Cancer
12:252-264). Exemplary anti-CTLA-4 antibodies include ipilimumab
(Bristol-Myers Squibb) and tremelimumab (Pfizer). Ipilimumab has
recently received FDA approval for treatment of metastatic melanoma
(Wada et al., 2013, J Transl Med 11:89).
[0650] Lymphocyte activation gene-3 (LAG-3), also known as CD223,
is another immune checkpoint protein. LAG-3 has been associated
with the inhibition of lymphocyte activity and in some cases the
induction of lymphocyte anergy. LAG-3 is expressed on various cells
in the immune system including B cells, NK cells, and dendritic
cells. LAG-3 is a natural ligand for the MHC class II receptor,
which is substantially expressed on melanoma-infiltrating T cells
including those endowed with potent immune-suppressive activity.
Exemplary anti-LAG-3 antibodies include BMS-986016 (Bristol-Myers
Squib), which is a monoclonal antibody that targets LAG-3. IMP701
(Immutep) is an antagonist LAG-3 antibody and IMP731 (Immutep and
GlaxoSmithKline) is a depleting LAG-3 antibody. Other LAG-3
inhibitors include IMP321 (Immutep), which is a recombinant fusion
protein of a soluble portion of LAG-3 and Ig that binds to MHC
class II molecules and activates antigen presenting cells (APC).
Other antibodies are described, e.g., in WO2010/019570 and US
2015/0259420
[0651] T-cell immunoglobulin domain and mucin domain-3 (TIM-3),
initially identified on activated Th1 cells, has been shown to be a
negative regulator of the immune response. Blockade of TIM-3
promotes T-cell mediated anti-tumor immunity and has anti-tumor
activity in a range of mouse tumor models. Combinations of TIM-3
blockade with other immunotherapeutic agents such as TSR-042,
anti-CD137 antibodies and others, can be additive or synergistic in
increasing anti-tumor effects. TIM-3 expression has been associated
with a number of different tumor types including melanoma, NSCLC
and renal cancer, and additionally, expression of intratumoral
TIM-3 has been shown to correlate with poor prognosis across a
range of tumor types including NSCLC, cervical, and gastric
cancers. Blockade of TIM-3 is also of interest in promoting
increased immunity to a number of chronic viral diseases. TIM-3 has
also been shown to interact with a number of ligands including
galectin-9, phosphatidylserine and HMGB1, although which of these,
if any, are relevant in regulation of anti-tumor responses is not
clear at present. In some embodiments, antibodies, antibody
fragments, small molecules, or peptide inhibitors that target TIM-3
can bind to the IgV domain of TIM-3 to inhibit interaction with its
ligands. Exemplary antibodies and peptides that inhibit TIM-3 are
described in US 2015/0218274, WO2013/006490 and US 2010/0247521.
Other anti-TIM-3 antibodies include humanized versions of RMT3-23
(Ngiow et al., 2011, Cancer Res, 71:3540-3551), and clone 8B.2C12
(Monney et al., 2002, Nature, 415:536-541). Bi-specific antibodies
that inhibit TIM-3 and PD-1 are described in US 2013/0156774.
[0652] In some embodiments, the additional agent is a CEACAM
inhibitor (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5 inhibitor). In
some embodiments, the inhibitor of CEACAM is an anti-CEACAM
antibody molecule. Exemplary anti-CEACAM-1 antibodies are described
in WO 2010/125571, WO 2013/082366 WO 2014/059251 and WO
2014/022332, e.g., a monoclonal antibody 34B1, 26H7, and 5F4; or a
recombinant form thereof, as described in, e.g., US 2004/0047858,
U.S. Pat. No. 7,132,255 and WO 99/052552. In some embodiments, the
anti-CEACAM antibody binds to CEACAM-5 as described in, e.g., Zheng
et al. PLoS One. (2011) 6(6): e21146), or cross-reacts with
CEACAM-1 and CEACAM-5 as described in, e.g., WO 2013/054331 and US
2014/0271618.
[0653] 4-1BB, also known as CD137, is transmembrane glycoprotein
belonging to the TNFR superfamily. 4-1BB receptors are present on
activated T cells and B cells and monocytes. An exemplary
anti-4-1BB antibody is urelumab (BMS-663513), which has potential
immunostimulatory and antineoplastic activities.
[0654] Tumor necrosis factor receptor superfamily, member 4
(TNFRSF4), also known as OX40 and CD134, is another member of the
TNFR superfamily. OX40 is not constitutively expressed on resting
naive T cells and acts as a secondary co-stimulatory immune
checkpoint molecule. Exemplary anti-OX40 antibodies are MEDI6469
and MOXR0916 (RG7888, Genentech).
[0655] In some embodiments, the additional agent includes a
molecule that decreases the regulatory T cell (Treg) population.
Methods that decrease the number of (e.g., deplete) Treg cells are
known and include, e.g., CD25 depletion, cyclophosphamide
administration, and modulating Glucocorticoid-induced TNFR family
related gene (GITR) function. GITR is a member of the TNFR
superfamily that is upregulated on activated T cells, which
enhances the immune system. Reducing the number of Treg cells in a
subject prior to apheresis or prior to administration of engineered
cells, e.g., CAR-expressing cells, can reduce the number of
unwanted immune cells (e.g., Tregs) in the tumor microenvironment
and reduces the subject's risk of relapse. In some embodiments, the
additional agent includes a molecule targeting GITR and/or
modulating GITR functions, such as a GITR agonist and/or a GITR
antibody that depletes regulatory T cells (Tregs). In some
embodiments, the additional agent includes cyclophosphamide. In
some embodiments, the GITR binding molecule and/or molecule
modulating GITR function (e.g., GITR agonist and/or Treg depleting
GITR antibodies) is administered prior to the engineered cells,
e.g., CAR-expressing cells. For example, in some embodiments, the
GITR agonist can be administered prior to apheresis of the cells.
In some embodiments, cyclophosphamide is administered to the
subject prior to administration (e.g., infusion or re-infusion) of
the engineered cells, e.g., CAR-expressing cells or prior to
apheresis of the cells. In some embodiments, cyclophosphamide and
an anti-GITR antibody are administered to the subject prior to
administration (e.g., infusion or re-infusion) of the engineered
cells, e.g., CAR-expressing cells or prior to apheresis of the
cells.
[0656] In some embodiments, the additional agent is a GITR agonist.
Exemplary GITR agonists include, e.g., GITR fusion proteins and
anti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as,
e.g., a GITR fusion protein described in U.S. Pat. No. 6,111,090,
European Patent No. 090505B 1, U.S. Pat. No. 8,586,023, PCT
Publication Nos.: WO 2010/003118 and 2011/090754, or an anti-GITR
antibody described, e.g., in U.S. Pat. No. 7,025,962, European
Patent No. 1947183B 1, U.S. Pat. Nos. 7,812,135, 8,388,967,
8,591,886, European Patent No. EP 1866339, WO 2011/028683, WO
2013/039954, WO2005/007190, WO 2007/133822, WO2005/055808, WO
99/40196, WO 2001/03720, WO99/20758, WO2006/083289, WO 2005/115451,
U.S. Pat. No. 7,618,632, and WO 2011/051726. An exemplary anti-GITR
antibody is TRX518.
[0657] In some embodiments, the additional agent enhances tumor
infiltration or transmigration of the administered cells, e.g.,
CAR-expressing cells. For example, in some embodiments, the
additional agent stimulates CD40, such as CD40L, e.g., recombinant
human CD40L. Cluster of differentiation 40 (CD40) is also a member
of the TNFR superfamily. CD40 is a costimulatory protein found on
antigen-presenting cells and mediates a broad variety of immune and
inflammatory responses. CD40 is also expressed on some
malignancies, where it promotes proliferation. Exemplary anti-CD40
antibodies are dacetuzumab (SGN-40), lucatumumab (Novartis,
antagonist), SEA-CD40 (Seattle Genetics), and CP-870,893. In some
embodiments, the additional agent that enhances tumor infiltration
includes tyrosine kinase inhibitor sunitnib, heparanase, and/or
chemokine receptors such as CCR2, CCR4, and CCR7.
[0658] In some embodiments, the additional agent is a structural or
functional analog or derivative of thalidomide and/or an inhibitor
of E3 ubiquitin ligase. In some embodiments, the immunomodulatory
agent binds to cereblon (CRBN). In some embodiments, the
immunomodulatory agent binds to the CRBN E3 ubiquitin-ligase
complex. In some embodiments, the immunomodulatory agent binds to
CRBN and the CRBN E3 ubiquitin-ligase complex. In some embodiments,
the immunomodulatory agent up-regulates the protein or gene
expression of CRBN. In some aspects, CRBN is the substrate adaptor
for the CRL4.sup.CRBN E3 ubiquitin ligase, and modulates the
specificity of the enzyme. In some embodiments, binding to CRB or
the CRBN E3 ubiquitin ligase complex inhibits E3 ubiquitin ligase
activity. In some embodiments, the immunomodulatory agent induces
the ubiquitination of KZF1 (Ikaros) and IKZF3 (Aiolos) and/or
induces degradation of IKZF1 (Ikaros) and IKZF3 (Aiolos). In some
embodiments, the immunomodulatory agent induces the ubiquitination
of casein kinase 1A1 (CK1.alpha.) by the CRL4.sup.CRBN E3 ubiquitin
ligase. In some embodiments, the ubiquitination of CK1.alpha.
results in CK1.alpha. degradation.
[0659] In some embodiments, the additional agent is an inhibitor of
the Ikaros (IKZF1) transcription factor. In some embodiments, the
additional agent enhances ubiquitination of Ikaros. In some
embodiments, the additional agent enhances the degradation of
Ikaros. In some embodiments, the additional agent down-regulates
the protein or gene expression of Ikaros. In some embodiments,
administration of the additional agent causes a decrease in Ikaros
protein levels.
[0660] In some embodiments, the additional agent is an inhibitor of
the Aiolos (IKZF3) transcription factor. In some embodiments, the
additional agent enhances ubiquitination of Aiolos. In some
embodiments, the additional agent enhances the degradation of
Aiolos. In some embodiments, the additional agent down-regulates
the protein or gene expression of Aiolos. In some embodiments,
administration of the additional agent causes a decrease in Aiolos
protein levels.
[0661] In some embodiments, the additional agent is an inhibitor of
both the Ikaros (IKZF1) and Aiolos (IKZF3) transcription factors.
In some embodiments, the additional agent enhances ubiquitination
of both Ikaros and Aiolos. In some embodiments, the additional
agent enhances the degradation of both Ikaros and Aiolos. In some
embodiments, the additional agent enhances ubiquitination and
degradation of both Ikaros and Aiolos. In some embodiments,
administration of the additional agent causes both Aiolos protein
levels and Ikaros protein levels to decrease.
[0662] In some embodiments, the additional agent is a selective
cytokine inhibitory drug (SelCID). In some embodiments, the
additional agent inhibits the activity of phosphodiesterase-4
(PDE4). In some embodiments, the additional agent suppresses the
enzymatic activity of the CDCl25 phosphatases. In some embodiments,
the additional agent alters the intracellular trafficking of CDCl25
phosphatases.
[0663] In some embodiments, the additional agent is thalidomide
(2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione) or an
analog or derivative of thalidomide. In certain embodiments, a
thalidomide derivative includes structural variants of thalidomide
that have a similar biological activity. Exemplary thalidomide
derivatives include, but are not limited to lenalidomide
(REVLIMMUNOMODULATORY COMPOUND.TM.; Celgene Corporation),
pomalidomide (also known as ACTIMMUNOMODULATORY COMPOUND.TM. or
POMALYST.TM. (Celgene Corporation)), CC-1088, CDC-501, and CDC-801,
and the compounds disclosed in U.S. Pat. Nos. 5,712,291; 7,320,991;
and 8,716,315; U.S. Appl. No. 2016/0313300; and PCT Pub. Nos. WO
2002/068414 and WO 2008/154252.
[0664] In some embodiments, the additional agent is 1-oxo- and 1,3
dioxo-2-(2,6-dioxopiperldin-3-yl) isoindolines substituted with
amino in the benzo ring as described in U.S. Pat. No. 5,635,517
which is incorporated herein by reference.
[0665] In some embodiments, the additional agent is a compound of
the following formula:
##STR00001##
[0666] wherein one of X and Y is --C(O)-- and the other of X and Y
is --C(O)-- or --CH.sub.2--, and R.sup.5 is hydrogen or lower
alkyl, or a pharmaceutically acceptable salt thereof. In some
embodiments, X is --C(O)-- and Y is --CH.sub.2--. In some
embodiments, both X and Y are --C(O)--. In some embodiments,
R.sup.5 is hydrogen. In other embodiments, R.sup.5 is methyl.
[0667] In some embodiments, the additional agent is a compound that
belongs to a class of substituted 2-(2,
6-dioxopiperidin-3-yl)phthalate immunomodulatory compounds and
substituted 2-(2,6-dioxopiperldin-3-yl)-1-oxoisoindoles, such as
those described in U.S. Pat. Nos. 6,281,230; 6,316,471; 6,335,349;
and 6,476,052, and International Patent Application No.
PCT/US97/13375 (International Publication No. WO 98/03502), each of
which is incorporated herein by reference.
[0668] In some embodiments, the additional agent is a compound of
the following formula:
##STR00002##
[0669] wherein
[0670] one of X and Y is --C(O)-- and the other of X and Y is
--C(O)-- or --CH.sub.2--;
[0671] (1) each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
independently halo, alkyl of 1 to 4 carbon atoms, or alkoxy or 1 to
4 carbon atoms, or
[0672] (2) one of R.sup.1, R.sup.3, R.sup.4, and R.sup.5 is
--NHR.sup.a and the remaining of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is are hydrogen, wherein R.sup.a is hydrogen or alkyl of 1
to 8 carbon atoms;
[0673] R.sup.5 is hydrogen or alkyl of 1 to 8 carbon atoms, benzyl,
or halo;
[0674] provided that R.sup.5 is other than hydrogen if X and Y are
--C(O)-- and (i) each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is
fluoro; or (ii) one of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is
amino;
[0675] or a pharmaceutically acceptable salt thereof.
[0676] In some embodiments, the additional agent is a compound that
belongs to a class of isoindole-immunomodulatory compounds
disclosed in U.S. Pat. No. 7,091,353, U.S. Patent Publication No.
2003/0045552, and International Application No. PCT/US01/50401
(International Publication No. WO02/059106), each of which are
incorporated herein by reference. For example, in some embodiments,
the additional agent is
[2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethy-
l]-amide;
(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol--
4-ylmethyl)-carbamic acid tert-butyl ester;
4-(aminomethyl)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione;
N-(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmet-
hyl)-acetamide;
N-{(2-(2,6-dioxo(3-piperidyl)-1,3-dioxoisoindolin-4-yl)methyl}cyclopropyl-
-carboxamide;
2-chloro-N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}a-
cetamide;
N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-3-pyridy-
lcarboxamide;
3-{1-oxo-4-(benzylamino)isoindolin-2-yl}piperidine-2,6-dione;
2-(2,6-dioxo(3-piperidyl))-4-(benzylamino)isoindoline-1,3-dione;
N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}propanamid-
e;
N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-3-pyrid-
ylcarboxamide;
N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}heptanamid-
e;
N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-2-furyl-
carboxamide;
{N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)carbamoyl}methyl
acetate;
N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)pentanami-
de;
N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-2-thienylcarbo-
xamide;
N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(bu-
tylamino)carboxamide;
N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(octylamin-
o)carboxamide; or
N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(benzylami-
no)carboxamide.
[0677] In some embodiments, the additional agent is a compound that
belongs to a class of isoindole-immunomodulatory compounds
disclosed in U.S. Patent Application Publication Nos. 2002/0045643,
International Publication No. WO 98/54170, and U.S. Pat. No.
6,395,754, each of which is incorporated herein by reference. In
some embodiments, the additional agent is a tetra substituted
2-(2,6-dioxopiperdin-3-yl)-1-oxoisoindolines described in U.S. Pat.
No. 5,798,368, which is incorporated herein by reference. In some
embodiments, the additional agent is 1-oxo and
1,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines disclosed in
U.S. Pat. No. 6,403,613, which is incorporated herein by reference.
In some embodiments the additional agent is a 1-oxo or
1,3-dioxoisoindoline substituted in the 4- or 5-position of the
indoline ring as described in U.S. Pat. Nos. 6,380,239 and
7,244,759, both of which are incorporated herein by reference.
[0678] In some embodiments, the additional agent is
2-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-carbamoyl-butyric
acid or
4-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-carbamoyl-butyric
acid. In some embodiments, the immunomodulatory compound is
4-carbamoyl-4-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoind-
ol-2-yl}-butyric acid,
4-carbamoyl-2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoind-
ol-2-yl}-butyric acid,
2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-4-p-
henylcarbamoyl-butyric acid, or
2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-pen-
tanedioic acid.
[0679] In some embodiments, the additional agent is a
isoindoline-1-one or isoindoline-1,3-dione substituted in the
2-position with 2,6-dioxo-3-hydroxypiperidin-5-yl as described in
U.S. Pat. No. 6,458,810, which is incorporated herein by reference.
In some embodiments, the immunomodulatory compound is
3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione,
or an enantiomer or a mixture of enantiomers thereof; or a
pharmaceutically acceptable salt, solvate, hydrate, co-crystal,
clathrate, or polymorph thereof. In some embodiments, the
immunomodulatory compound is
3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]--
piperidine-2,6-dione.
[0680] In some embodiments, the additional agent is as described in
Oshima, K. et al., Nihon Rinsho., 72(6):1130-5 (2014); Millrine, D.
et al., Trends Mol Med., 23(4):348-364 (2017); and Collins, et al.,
Biochem J., 474(7):1127-1147 (2017).
[0681] In some embodiments, the additional agent includes
thalidomide drugs or analogs thereof and/or derivatives thereof,
such as lenalidomide, pomalidomide or apremilast. See, e.g.,
Bertilaccio et at, Blood (2013) 122:4171, Otahal et al.,
Oncoimmunology (2016) 5(4):e1115940; Fecteau et al., Blood (2014)
124(10):1637-1644 and Kuramitsu et al., Cancer Gene Therapy (2015)
22:487-495). Lenalidomide
((RS)-3-(4-Amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione;
also known as Revlimid) is a synthetic derivative of thalidomide,
and has multiple immunomodulatory effects, including enforcement of
immune synapse formation between T cell and antigen presenting
cells (APCs). In some embodiments, the additional agent is
lenalidomide, pomalidomide, avadomide, a stereoisomer of
lenalidomide, pomalidomide, avadomide or a pharmaceutically
acceptable salt, solvate, hydrate, co-crystal, clathrate, or
polymorph thereof. In some embodiments, the immunomodulatory
compound is lenalidomide, a stereoisomer of lenalidomide or a
pharmaceutically acceptable salt, solvate, hydrate, co-crystal,
clathrate, or polymorph thereof. In some embodiments, the
immunomodulatory compound is lenalidomide, or
((RS)-3-(4-Amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione)-
.
[0682] In certain embodiments, the lesion is disrupted by
administering the thalidomide derivative lenalidomide,
((RS)-3-(4-Amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione)
to the subject. Lenalidomide is FDA approved for the treatment of
multiple myeloma, myelodysplastic syndrome associated with deletion
5q, and most recently in relapsed/refractory mantle-cell lymphoma
(MCL). Lenalidomide generally is a synthetic derivative of
thalidomide, and is currently understood to have multiple
immunomodulatory effects, including enforcement of immune synapse
formation between T cell and antigen presenting cells (APCs). For
example, in some cases, lenalidomide modulates T cell responses and
results in increased interleukin (IL)-2 production in CD4.sup.+ and
CD8.sup.+ T cells, induces the shift of T helper (Th) responses
from Th2 to Th1, inhibits expansion of regulatory subset of T cells
(Tregs), and improves functioning of immunological synapses in
follicular lymphoma and chronic lymphocytic leukemia (CLL) (Otahal
et al., Oncoimmunology (2016) 5(4):e1115940). Lenalidomide also has
direct tumoricidal activity in patients with multiple myeloma (MM)
and directly and indirectly modulates survival of CLL tumor cells
by affecting supportive cells, such as nurse-like cells found in
the microenvironment of lymphoid tissues. Lenalidomide also can
enhance T-cell proliferation and interferon-.gamma. production in
response to activation of T cells via CD3 ligation or dendritic
cell-mediated activation. In addition, lenalidomide is thought to
decrease proliferation of pro-inflammatory cytokines including
TNF-.alpha., IL-1, IL-6, and IL-12 and enhance antibody-dependent
cellular cytotoxicity (ADCC) via increased NK cell activation.
Lenalidomide can also induce malignant B cells to express higher
levels of immunostimulatory molecules such as CD80, CD86, HLA-DR,
CD95, and CD40 (Fecteau et al., Blood (2014) 124(10):1637-1644).
Cereblon, an E3 ubiquitin ligase, was identified as the primary
target for thalidomide-induced teratogenesis (Ito et al., T.,
(2010) Science 327: 1345-1350). Lenalidomide also targets cereblon
and it has been shown that this leads to the reduction of c-Myc and
IRF4 expression while also increasing expression of p21 that leads
to G1 cell-cycle arrest (Lopez-Girona et al., (2012) Leukemia 26:
2326-2335).
[0683] In some embodiments, the additional agent is a B-cell
inhibitor. In some embodiments, the additional agent is one or more
B-cell inhibitors selected from among inhibitors of CD10, CD19,
CD20, CD22, CD34, CD123, CD79a, CD79b, CD179b, FLT-3, or ROR1, or a
combination thereof. In some embodiments, the B-cell inhibitor is
an antibody (e.g., a mono- or bispecific antibody) or an antigen
binding fragment thereof. In some embodiments, the additional agent
is an engineered cell expressing recombinant receptors that target
B-cell targets, e.g., CD10, CD19, CD20, CD22, CD34, CD123, CD79a,
CD79b, CD179b, FLT-3, or ROR1.
[0684] In some embodiments, the additional agent is a CD20
inhibitor, e.g., an anti-CD20 antibody (e.g., an anti-CD20 mono- or
bi-specific antibody) or a fragment thereof. Exemplary anti-CD20
antibodies include but are not limited to rituximab, ofatumumab,
ocrelizumab (also known as GA101 or R05072759), veltuzumab,
obinutuzumab, TRU-015 (Trubion Pharmaceuticals), ocaratuzumab (also
known as AME-133v or ocaratuzumab), and Pro131921 (Genentech). See,
e.g., Lim et al. Haematologica. (2010) 95(1):135-43. In some
embodiments, the anti-CD20 antibody comprises rituximab. Rituximab
is a chimeric mouse/human monoclonal antibody IgG1 kappa that binds
to CD20 and causes cytolysis of a CD20 expressing cell. In some
embodiments, the additional agent includes rituximab. In some
embodiments, the CD20 inhibitor is a small molecule.
[0685] In some embodiments, the additional agent is a CD22
inhibitor, e.g., an anti-CD22 antibody (e.g., an anti-CD22 mono- or
bi-specific antibody) or a fragment thereof. Exemplary anti-CD22
antibodies include epratuzumab and RFB4. In some embodiments, the
CD22 inhibitor is a small molecule. In some embodiments, the
antibody is a monospecific antibody, optionally conjugated to a
second agent such as a chemotherapeutic agent. For instance, in
some embodiments, the antibody is an anti-CD22 monoclonal
antibody-MMAE conjugate (e.g., DCDT2980S). In some embodiments, the
antibody is an scFv of an anti-CD22 antibody, e.g., an scFv of
antibody RFB4. In some embodiments, the scFv is fused to all of or
a fragment of Pseudomonas exotoxin-A (e.g., BL22). In some
embodiments, the scFv is fused to all of or a fragment of (e.g., a
38 kDa fragment of) Pseudomonas exotoxin-A (e.g., moxetumomab
pasudotox). In some embodiments, the anti-CD22 antibody is an
anti-CD19/CD22 bispecific antibody, optionally conjugated to a
toxin. For instance, in some embodiments, the anti-CD22 antibody
comprises an anti-CD19/CD22 bispecific portion, (e.g., two scFv
ligands, recognizing human CD19 and CD22) optionally linked to all
of or a portion of diphtheria toxin (DT), e.g., first 389 amino
acids of diphtheria toxin (DT), DT 390, e.g., a ligand-directed
toxin such as DT2219ARL). In some embodiments, the bispecific
portion (e.g., anti-CD 19/anti-CD22) is linked to a toxin such as
deglycosylated ricin A chain (e.g., Combotox).
[0686] In some embodiments, the immunomodulatory agent is a
cytokine. In some embodiments, the immunomodulatory agent is a
cytokine or is an agent that induces increased expression of a
cytokine in the tumor microenvironment. Cytokines have important
functions related to T cell expansion, differentiation, survival,
and homeostasis. Cytokines that can be administered to the subject
receiving the binding molecules (e.g., ROR1-binding molecules),
recombinant receptors, cells and/or compositions provided herein
include one or more of IL-2, IL-4, IL-7, IL-9, IL-15, IL-18, and
IL-21. In some embodiments, the cytokine administered is IL-7,
IL-15, or IL-21, or a combination thereof. In some embodiments,
administration of the cytokine to the subject that has sub-optimal
response to the administration of the engineered cells, e.g.,
CAR-expressing cells improves efficacy and/or anti-tumor activity
of the administered cells, e.g., CAR-expressing cells.
[0687] By "cytokine" is meant a generic term for proteins released
by one cell population that act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormones such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-alpha and -beta;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-beta; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I
and -II; erythropoietin (EPO); osteoinductive factors; interferons
such as interferon-alpha, beta, and -gamma; colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor
necrosis factor such as TNF-alpha or TNF-beta; and other
polypeptide factors including LIF and kit ligand (KL). As used
herein, the term cytokine includes proteins from natural sources or
from recombinant cell culture, and biologically active equivalents
of the native sequence cytokines. For example, the immunomodulatory
agent is a cytokine and the cytokine is IL-4, TNF-.alpha., GM-CSF
or IL-2.
[0688] In some embodiments, the additional agent includes an
interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor
alpha (IL-15Ra) polypeptide, or combination thereof, e.g., hetIL-15
(Admune Therapeutics, LLC). hetIL-15 is a heterodimeric
non-covalent complex of IL-15 and IL-15R.alpha.. hetIL-15 is
described in, e.g., U.S. Pat. No. 8,124,084, U.S. 2012/0177598,
U.S. 2009/0082299, U.S. 2012/0141413, and U.S. 2011/0081311. In
some embodiments, the immunomodulatory agent can contain one or
more cytokines. For example, the interleukin can include leukocyte
interleukin injection (Multikine), which is a combination of
natural cytokines. In some embodiments, the immunomodulatory agent
is a Toll-like receptor (TLR) agonist, an adjuvant or a
cytokine.
[0689] In some embodiments, the additional agent is an agent that
ameliorates or neutralizes one or more toxicities or side effects
associated with the cell therapy. In some embodiments, the
additional agent is selected from among a steroid (e.g.,
corticosteroid), an inhibitor of TNF.alpha., and an inhibitor of
IL-6. An example of a TNF.alpha. inhibitor is an anti-TNF.alpha.
antibody molecule such as, infliximab, adalimumab, certolizumab
pegol, and golimumab. Another example of a TNF.alpha. inhibitor is
a fusion protein such as entanercept. Small molecule inhibitors of
TNF.alpha. include, but are not limited to, xanthine derivatives
(e.g. pentoxifylline) and bupropion. An example of an IL-6
inhibitor is an anti-IL-6 antibody molecule such as tocilizumab,
sarilumab, elsilimomab, CNTO 328, ALD518/BMS-945429, CNTO 136,
CPSI-2364, CDP6038, VX30, ARGX-109, FE301, and FM101. In some
embodiments, the anti-IL-6 antibody molecule is tocilizumab. In
some embodiments, the additional agent is an IL-1R inhibitor, such
as anakinra
[0690] In some embodiments, the additional agent is a modulator of
adenosine levels and/or an adenosine pathway component. Adenosine
can function as an immunomodulatory agent in the body. For example,
adenosine and some adenosine analogs that non-selectively activate
adenosine receptor subtypes decrease neutrophil production of
inflammatory oxidative products (Cronstein et al., Ann. N.Y. Acad.
Sci. 451:291, 1985; Roberts et al., Biochem. J., 227:669, 1985;
Schrier et al., J. Immunol. 137:3284, 1986; Cronstein et al.,
Clinical Immunol. Immunopath. 42:76, 1987). In some cases,
concentration of extracellular adenosine or adenosine analogs can
increase in specific environments, e.g., tumor microenvironment
(TME). In some cases, adenosine or adenosine analog signaling
depends on hypoxia or factors involved in hypoxia or its
regulation, e.g., hypoxia inducible factor (HIF). In some
embodiments, increase in adenosine signaling can increase in
intracellular cAMP and cAMP-dependent protein kinase that results
in inhibition of proinflammatory cytokine production, and can lead
to the synthesis of immunosuppressive molecules and development of
Tregs (Sitkovsky et al., Cancer Immunol Res (2014) 2(7):598-605).
In some embodiments, the additional agent can reduce or reverse
immunosuppressive effects of adenosine, adenosine analogs and/or
adenosine signaling. In some embodiments, the additional agent can
reduce or reverse hypoxia-driven A2-adenosinergic T cell
immunosuppression. In some embodiments, the additional agent is
selected from among antagonists of adenosine receptors,
extracellular adenosine-degrading agents, inhibitors of adenosine
generation by CD39/CD73 ectoenzymes, and inhibitors of
hypoxia-HIF-la signaling. In some embodiments, the additional agent
is an adenosine receptor antagonist or agonist.
[0691] Inhibition or reduction of extracellular adenosine or the
adenosine receptor by virtue of an inhibitor of extracellular
adenosine (such as an agent that prevents the formation of,
degrades, renders inactive, and/or decreases extracellular
adenosine), and/or an adenosine receptor inhibitor (such as an
adenosine receptor antagonist) can enhance immune response, such as
a macrophage, neutrophil, granulocyte, dendritic cell, T- and/or B
cell-mediated response. In addition, inhibitors of the Gs protein
mediated cAMP dependent intracellular pathway and inhibitors of the
adenosine receptor-triggered Gi protein mediated intracellular
pathways, can also increase acute and chronic inflammation.
[0692] In some embodiments, the additional agent is an adenosine
receptor antagonist or agonist, e.g., an antagonist or agonist of
one or more of the adenosine receptors A2a, A2b, A1, and A3. A1 and
A3 inhibit, and A2a and A2b stimulate, respectively, adenylate
cyclase activity. Certain adenosine receptors, such as A2a, A2b,
and A3, can suppress or reduce the immune response during
inflammation. Thus, antagonizing immunosuppressive adenosine
receptors can augment, boost or enhance immune response, e.g.,
immune response from administered cells, e.g., CAR-expressing T
cells. In some embodiments, the additional agent inhibits the
production of extracellular adenosine and adenosine-triggered
signaling through adenosine receptors. For example, enhancement of
an immune response, local tissue inflammation, and targeted tissue
destruction can be enhanced by inhibiting or reducing the
adenosine-producing local tissue hypoxia; by degrading (or
rendering inactive) accumulated extracellular adenosine; by
preventing or decreasing expression of adenosine receptors on
immune cells; and/or by inhibiting/antagonizing signaling by
adenosine ligands through adenosine receptors.
[0693] An antagonist is any substance that tends to nullify the
action of another, as an agent that binds to a cell receptor
without eliciting a biological response. In some embodiments, the
antagonist is a chemical compound that is an antagonist for an
adenosine receptor, such as the A2a, A2b, or A3 receptor. In some
embodiments, the antagonist is a peptide, or a peptidomimetic, that
binds the adenosine receptor but does not trigger a Gi protein
dependent intracellular pathway. Exemplary antagonists are
described in U.S. Pat. Nos. 5,565,566; 5,545,627, 5,981,524;
5,861,405; 6,066,642; 6,326,390; 5,670,501; 6,117,998; 6,232,297;
5,786,360; 5,424,297; 6,313,131, 5,504,090; and 6,322,771.
[0694] In some embodiments, the additional agent is an A2 receptor
(A2R) antagonist, such as an A2a antagonist. Exemplary A2R
antagonists include KW6002 (istradefyline), SCH58261, caffeine,
paraxanthine, 3,7-dimethyl-1-propargylxanthine (DMPX),
8-(m-chlorostyryl) caffeine (CSC), MSX-2, MSX-3, MSX-4, CGS-15943,
ZM-241385, SCH-442416, preladenant, vipadenant (BII014), V2006,
ST-1535, SYN-115, PSB-1115, ZM241365, FSPTP, and an inhibitory
nucleic acid targeting A2R expression, e.g., siRNA or shRNA, or any
antibodies or antigen-binding fragment thereof that targets an A2R.
In some embodiments, the additional agent is an A2R antagonist
described in, e.g., Ohta et al., Proc Natl Acad Sci USA (2006)
103:13132-13137; Jin et al., Cancer Res. (2010) 70(6):2245-2255;
Leone et al., Computational and Structural Biotechnology Journal
(2015) 13:265-272; Beavis et al., Proc Natl Acad Sci USA (2013)
110:14711-14716; and Pinna, A., Expert Opin Investig Drugs (2009)
18:1619-1631; Sitkovsky et al., Cancer Immunol Res (2014)
2(7):598-605; U.S. Pat. Nos. 8,080,554; 8,716,301; US 20140056922;
WO2008/147482; U.S. Pat. No. 8,883,500; US 20140377240;
WO02/055083; U.S. Pat. Nos. 7,141,575; 7,405,219; 8,883,500;
8,450,329 and 8,987,279).
[0695] In some embodiments, the antagonist is an antisense
molecule, inhibitory nucleic acid molecule (e.g., small inhibitory
RNA (siRNA)) or catalytic nucleic acid molecule (e.g. a ribozyme)
that specifically binds mRNA encoding an adenosine receptor. In
some embodiments, the antisense molecule, inhibitory nucleic acid
molecule or catalytic nucleic acid molecule binds nucleic acids
encoding A2a, A2b, or A3. In some embodiments, an antisense
molecule, inhibitory nucleic acid molecule or catalytic nucleic
acid targets biochemical pathways downstream of the adenosine
receptor. For example, the antisense molecule or catalytic nucleic
acid can inhibit an enzyme involved in the Gs protein- or Gi
protein-dependent intracellular pathway. In some embodiments, the
additional agent includes dominant negative mutant form of an
adenosine receptor, such as A2a, A2b, or A3.
[0696] In some embodiments, the additional agent that inhibits
extracellular adenosine includes agents that render extracellular
adenosine non-functional (or decrease such function), such as a
substance that modifies the structure of adenosine to inhibit the
ability of adenosine to signal through adenosine receptors. In some
embodiments, the additional agent is an extracellular
adenosine-generating or adenosine-degrading enzyme, a modified form
thereof or a modulator thereof. For example, in some embodiments,
the additional agent is an enzyme (e.g. adenosine deaminase) or
another catalytic molecule that selectively binds and destroys the
adenosine, thereby abolishing or significantly decreasing the
ability of endogenously formed adenosine to signal through
adenosine receptors and terminate inflammation.
[0697] In some embodiments, the additional agent is an adenosine
deaminase (ADA) or a modified form thereof, e.g., recombinant ADA
and/or polyethylene glycol-modified ADA (ADA-PEG), which can
inhibit local tissue accumulation of extracellular adenosine.
ADA-PEG has been used in treatment of patients with ADA SCID
(Hershfield (1995) Hum Mutat. 5:107). In some embodiments, an agent
that inhibits extracellular adenosine includes agents that prevent
or decrease formation of extracellular adenosine, and/or prevent or
decrease the accumulation of extracellular adenosine, thereby
abolishing, or substantially decreasing, the immunosuppressive
effects of adenosine. In some embodiments, the additional agent
specifically inhibits enzymes and proteins that are involved in
regulation of synthesis and/or secretion of pro-inflammatory
molecules, including modulators of nuclear transcription factors.
Suppression of adenosine receptor expression or expression of the
Gs protein- or Gi protein-dependent intracellular pathway, or the
cAMP dependent intracellular pathway, can result in an
increase/enhancement of immune response.
[0698] In some embodiments, the additional agent can target
ectoenzymes that generate or produce extracellular adenosine. In
some embodiments, the additional agent targets CD39 and CD73
ectoenzymes, which function in tandem to generate extracellular
adenosine. CD39 (also called ectonucleoside triphosphate
diphosphohydrolase) converts extracellular ATP (or ADP) to 5'AMP.
Subsequently, CD73 (also called 5'nucleotidase) converts 5'AMP to
adenosine. The activity of CD39 is reversible by the actions of NDP
kinase and adenylate kinase, whereas the activity of CD73 is
irreversible. CD39 and CD73 are expressed on tumor stromal cells,
including endothelial cells and Tregs, and also on many cancer
cells. For example, the expression of CD39 and CD73 on endothelial
cells is increased under the hypoxic conditions of the tumor
microenvironment. Tumor hypoxia can result from inadequate blood
supply and disorganized tumor vasculature, impairing delivery of
oxygen (Carroll and Ashcroft (2005), Expert. Rev. Mol. Med.
7(6):1-16). Hypoxia also inhibits adenylate kinase (AK), which
converts adenosine to AMP, leading to very high extracellular
adenosine concentration. Thus, adenosine is released at high
concentrations in response to hypoxia, which is a condition that
frequently occurs the tumor microenvironment (TME), in or around
solid tumors. In some embodiments, the additional agent is one or
more of anti-CD39 antibody or antigen binding fragment thereof,
anti-CD73 antibody or antigen binding fragment thereof, e.g.,
MEDI9447 or TY/23, .alpha.-.beta.-methylene-adenosine diphosphate
(ADP), ARL 67156, POM-3, IPH52 (see, e.g., Allard et al. Clin
Cancer Res (2013) 19(20):5626-5635; Hausler et al., Am J Transl Res
(2014) 6(2):129-139; Zhang, B., Cancer Res. (2010)
70(16):6407-6411).
[0699] In some embodiments, the additional agent is an inhibitor of
hypoxia inducible factor 1 alpha (HIF-1a) signaling. Exemplary
inhibitors of HIF-la include digoxin, acriflavine, sirtuin-7 and
ganetespib.
[0700] In some embodiments, the additional agent includes a protein
tyrosine phosphatase inhibitor, e.g., a protein tyrosine
phosphatase inhibitor described herein. In some embodiments, the
protein tyrosine phosphatase inhibitor is an SHP-1 inhibitor, e.g.,
an SHP-1 inhibitor described herein, such as, e.g., sodium
stibogluconate. In some embodiments, the protein tyrosine
phosphatase inhibitor is an SHP-2 inhibitor, e.g., an SHP-2
inhibitor described herein.
[0701] In some embodiments, the additional agent is a kinase
inhibitor. Kinase inhibitors, such as a CDK4 kinase inhibitor, a
BTK kinase inhibitor, a MNK kinase inhibitor, or a DGK kinase
inhibitor, can regulate the constitutively active survival pathways
that exist in tumor cells and/or modulate the function of immune
cells. In some embodiments, the kinase inhibitor is a Bruton's
tyrosine kinase (BTK) inhibitor, e.g., ibrutinib. In some
embodiments, the kinase inhibitor is a
phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitor. In
some embodiments, the kinase inhibitor is a CDK4 inhibitor, e.g., a
CDK4/6 inhibitor. In some embodiments, the kinase inhibitor is an
mTOR inhibitor, such as, e.g., rapamycin, a rapamycin analog,
OSI-027. The mTOR inhibitor can be, e.g., an mTORC1 inhibitor
and/or an mTORC2 inhibitor, e.g., an mTORC1 inhibitor and/or mTORC2
inhibitor. In some embodiments, the kinase inhibitor is an MNK
inhibitor, or a dual PI3K/mTOR inhibitor. In some embodiments,
other exemplary kinase inhibitors include the AKT inhibitor
perifosine, the mTOR inhibitor temsirolimus, the Src kinase
inhibitors dasatinib and fostamatinib, the JAK2 inhibitors
pacritinib and ruxolitinib, the PKC.beta. inhibitors enzastaurin
and bryostatin, and the AAK inhibitor alisertib.
[0702] In some embodiments, the kinase inhibitor is a BTK inhibitor
selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560;
CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. In some
embodiments, the BTK inhibitor does not reduce or inhibit the
kinase activity of interleukin-2-inducible kinase (ITK), and is
selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224;
CC-292; ONO-4059; CNX-774; and LFM-A13.
[0703] In some embodiments, the kinase inhibitor is a BTK
inhibitor, e.g., ibrutinib
(1-[(3R)-3-[4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl]piperidin-1-yl]prop-2-en-1-one; also known as PCI-32765).
In some embodiments, the kinase inhibitor is a BTK inhibitor, e.g.,
ibrutinib (PCI-32765). In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12 or more cycles of ibrutinib are administered. In some
embodiments, the BTK inhibitor is a BTK inhibitor described in
International Application WO 2015/079417.
[0704] In some embodiments, the kinase inhibitor is a PI3K
inhibitor. PI3K is central to the PI3K/Akt/mTOR pathway involved in
cell cycle regulation and lymphoma survival. Exemplary PI3K
inhibitor includes idelalisib (PI3K.delta. inhibitor). In some
embodiments, the additional agent is idelalisib and rituximab.
[0705] In some embodiments, the additional agent is an inhibitor of
mammalian target of rapamycin (mTOR). In some embodiments, the
kinase inhibitor is an mTOR inhibitor selected from temsirolimus;
ridaforolimus (also known as AP23573 and MK8669); everolimus
(RAD001); rapamycin (AY22989); simapimod; AZD8055; PF04691502;
SF1126; and XL765. In some embodiments, the additional agent is an
inhibitor of mitogen-activated protein kinase (MAPK), such as
vemurafenib, dabrafenib, and trametinib.
[0706] In some embodiments, the additional agent is an agent that
regulates pro- or anti-apoptotic proteins. In some embodiments, the
additional agent includes a B-cell lymphoma 2 (BCL-2) inhibitor
(e.g., venetoclax, also called ABT-199 or GDC-0199; or ABT-737).
Venetoclax is a small molecule
(4-(4-{[2-(4-Chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piper-
azinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfon-
yl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide) that inhibits
the anti-apoptotic protein, BCL-2. Other agents that modulate pro-
or anti-apoptotic protein include BCL-2 inhibitor ABT-737,
navitoclax (ABT-263); Mcl-1 siRNA or Mcl-1 inhibitor retinoid
N-(4-hydroxyphenyl) retinamide (4-HPR) for maximal efficacy. In
some embodiments, the additional agent provides a pro-apoptotic
stimuli, such as recombinant tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL), which can activate the apoptosis
pathway by binding to TRAIL death receptors DR-4 and DR-5 on tumor
cell surface, or TRAIL-R2 agonistic antibodies.
[0707] In some embodiments, the additional agent includes an
indoleamine 2,3-dioxygenase (IDO) inhibitor. IDO is an enzyme that
catalyzes the degradation of the amino acid, L-tryptophan, to
kynurenine. Many cancers overexpress IDO, e.g., prostatic,
colorectal, pancreatic, cervical, gastric, ovarian, head, and lung
cancer. Plasmacytoid dendritic cells (pDCs), macrophages, and
dendritic cells (DCs) can express IDO. In some aspects, a decrease
in L-tryptophan (e.g., catalyzed by IDO) results in an
immunosuppressive milieu by inducing T-cell anergy and apoptosis.
Thus, in some aspects, an IDO inhibitor can enhance the efficacy of
the binding molecules (e.g., ROR1-binding molecules), recombinant
receptors, cells and/or compositions described herein, e.g., by
decreasing the suppression or death of the administered
CAR-expressing cell. Exemplary inhibitors of IDO include but are
not limited to 1-methyl-tryptophan, indoximod, and INCB024360
(epacadostat).
[0708] In some embodiments, the additional agent includes a
cytotoxic agent, e.g., CPX-351 (Celator Pharmaceuticals),
cytarabine, daunorubicin, vosaroxin (Sunesis Pharmaceuticals),
sapacitabine (Cyclacel Pharmaceuticals), idarubicin, or
mitoxantrone. In some embodiments, the additional agent includes a
hypomethylating agent, e.g., a DNA methyltransferase inhibitor,
e.g., azacitidine or decitabine.
[0709] In another embodiment, the additional therapy is a
transplantation, e.g., allogeneic stem cell transplant.
[0710] In some embodiments, the additional therapy is a
lymphodepleting therapy. In some embodiments, lymphodepletion is
performed on a subject, e.g., prior to administering engineered
cells, e.g., CAR-expressing cells. In some embodiments, the
lymphodepletion comprises administering one or more of melphalan,
Cytoxan, cyclophosphamide, and fludarabine. In some embodiments, a
lymphodepleting chemotherapy is administered to the subject prior
to, concurrently with, or after administration (e.g., infusion) of
engineered cells, e.g., CAR-expressing cells. In an example, the
lymphodepleting chemotherapy is administered to the subject prior
to administration of engineered cells, e.g., CAR-expressing
cells.
[0711] In some embodiments, the additional agent is an oncolytic
virus. In some embodiments, oncolytic viruses are capable of
selectively replicating in and triggering the death of or slowing
the growth of a cancer cell. In some cases, oncolytic viruses have
no effect or a minimal effect on non-cancer cells. An oncolytic
virus includes but is not limited to an oncolytic adenovirus,
oncolytic Herpes Simplex Viruses, oncolytic retrovirus, oncolytic
parvovirus, oncolytic vaccinia virus, oncolytic Sinbis virus,
oncolytic influenza virus, or oncolytic RNA virus (e.g., oncolytic
reovirus, oncolytic Newcastle Disease Virus (NDV), oncolytic
measles virus, or oncolytic vesicular stomatitis virus (VSV)).
[0712] Other exemplary combination therapy, treatment and/or agents
include anti-allergenic agents, anti-emetics, analgesics and
adjunct therapies. In some embodiments, the additional agent
includes cytoprotective agents, such as neuroprotectants,
free-radical scavengers, cardioprotectors, anthracycline
extravasation neutralizers and nutrients.
[0713] In some embodiments, an antibody used as an additional agent
is conjugated or otherwise bound to a therapeutic agent, e.g., a
chemotherapeutic agent (e.g., Cytoxan, fludarabine, histone
deacetylase inhibitor, demethylating agent, peptide vaccine,
anti-tumor antibiotic, tyrosine kinase inhibitor, alkylating agent,
anti-microtubule or anti-mitotic agent), anti-allergic agent,
anti-nausea agent (or anti-emetic), pain reliever, or
cytoprotective agent described herein. In some embodiments, the
additional agent is an antibody-drug conjugate.
[0714] In some embodiments, the additional agent can modulate,
inhibit or stimulate particular factors at the DNA, RNA or protein
levels, to enhance or boost the efficacy of the binding molecules
(e.g., ROR1-binding molecules), recombinant receptors, cells and/or
compositions provided herein. In some embodiments, the additional
agent can modulate the factors at the nucleic acid level, e.g., DNA
or RNA, within the administered cells, e.g., cells engineered to
express recombinant receptors, e.g., CAR. In some embodiments, an
inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a
dsRNA, e.g., an siRNA or shRNA, or a clustered regularly
interspaced short palindromic repeats (CRISPR), a
transcription-activator like effector nuclease (TALEN), or a zinc
finger endonuclease (ZFN), can be used to inhibit expression of an
inhibitory molecule in the engineered cell, e.g., CAR-expressing
cell. In some embodiments the inhibitor is an shRNA. In some
embodiments, the inhibitory molecule is inhibited within the
engineered cell, e.g., CAR-expressing cell. In some embodiments, a
nucleic acid molecule that encodes a dsRNA molecule that inhibits
expression of the molecule that modulates or regulates, e.g.,
inhibits, T-cell function is operably linked to a promoter, e.g., a
HI- or a U6-derived promoter such that the dsRNA molecule that
inhibits expression of the inhibitory molecule is expressed within
the engineered cell, e.g., CAR-expressing cell. See, e.g.,
Brummelkamp T R, et al. (2002) Science 296: 550-553; Miyagishi M,
et al. (2002) Nat. Biotechnol. 19: 497-500.
[0715] In some embodiments, the additional agent is capable of
disrupting the gene encoding an inhibitory molecule, such as any
immune checkpoint inhibitors described herein. In some embodiments,
disruption is by deletion, e.g., deletion of an entire gene, exon,
or region, and/or replacement with an exogenous sequence, and/or by
mutation, e.g., frameshift or missense mutation, within the gene,
typically within an exon of the gene. In some embodiments, the
disruption results in a premature stop codon being incorporated
into the gene, such that the inhibitory molecule is not expressed
or is not expressed in a form that is capable of being expressed on
the cells surface and/or capable of mediating cell signaling. The
disruption is generally carried out at the DNA level. The
disruption generally is permanent, irreversible, or not
transient.
[0716] In some aspects, the disruption is carried out by gene
editing, such as using a DNA binding protein or DNA-binding nucleic
acid, which specifically binds to or hybridizes to the gene at a
region targeted for disruption. In some aspects, the protein or
nucleic acid is coupled to or complexed with a nuclease, such as in
a chimeric or fusion protein. For example, in some embodiments, the
disruption is effected using a fusion comprising a DNA-targeting
protein and a nuclease, such as a Zinc Finger Nuclease (ZFN) or
TAL-effector nuclease (TALEN), or an RNA-guided nuclease such as a
clustered regularly interspersed short palindromic nucleic acid
(CRISPR)-Cas system, such as CRISPR-Cas9 system, specific for the
gene being disrupted. In some embodiments, methods of producing or
generating genetically engineered cells, e.g., CAR-expressing
cells, include introducing into a population of cells nucleic acid
molecules encoding a genetically engineered antigen receptor (e.g.
CAR) and nucleic acid molecules encoding an agent targeting an
inhibitory molecule that is a gene editing nuclease, such as a
fusion of a DNA-targeting protein and a nuclease such as a ZFN or a
TALEN, or an RNA-guided nuclease such as of the CRISPR-Cas9 system,
specific for an inhibitory molecule.
[0717] Any of the additional agents described herein can be
prepared and administered as combination therapy with the
ROR1-binding molecule (e.g., antibody), immunoconjugate,
recombinant receptor (e.g., chimeric antigen receptor) and/or
engineered cells expressing said molecules (e.g., recombinant
receptor) described herein, such as in pharmaceutical compositions
comprising one or more agents of the combination therapy and a
pharmaceutically acceptable carrier, such as any described herein.
In some embodiments, the ROR1-binding molecule (e.g., antibody),
immunoconjugate, recombinant receptor (e.g., chimeric antigen
receptor), engineered cells expressing said molecules (e.g.,
recombinant receptor), plurality of engineered cells expressing
said molecules (e.g., recombinant receptor) can be administered
simultaneously, concurrently or sequentially, in any order with the
additional agents, therapy or treatment, wherein such
administration provides therapeutically effective levels each of
the agents in the body of the subject. The agents can be
co-administered with the binding molecules (e.g., ROR1-binding
molecules), recombinant receptors, cells and/or compositions
described herein, for example, as part of the same pharmaceutical
composition or using the same method of delivery. In some
embodiments, the additional agent is incubated with the engineered
cell, e.g., CAR-expressing cells, prior to administration of the
cells.
[0718] In some examples, the one or more additional agents are
administered subsequent to or prior to the administration of the
binding molecules (e.g., ROR1-binding molecules), recombinant
receptors, cells and/or compositions described herein, separated by
a selected time period. In some examples, the time period is 1 day,
2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1
month, 2 months, or 3 months. In some examples, the one or more
additional agents are administered multiple times and/or the
binding molecules (e.g., ROR1-binding molecules), recombinant
receptors, cells and/or compositions described herein, is
administered multiple times. For example, in some embodiments, the
additional agent is administered prior to the binding molecules
(e.g., ROR1-binding molecules), recombinant receptors, cells and/or
compositions described herein, e.g., two weeks, 12 days, 10 days, 8
days, one week, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day
before the administration. For example, in some embodiments, the
additional agent is administered after the binding molecules (e.g.,
ROR1-binding molecules), recombinant receptors, cells and/or
compositions described herein, e.g., two weeks, 12 days, 10 days, 8
days, one week, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day
after the administration.
[0719] The dose of the additional agent can be any therapeutically
effective amount, e.g., any dose amount described herein, and the
appropriate dosage of the additional agent may depend on the type
of disease to be treated, the type, dose and/or frequency of the
binding molecule, recombinant receptor, cell and/or composition
administered, the severity and course of the disease, whether the
binding molecule, recombinant receptor, cell and/or composition is
administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the binding
molecule, recombinant receptor, cell and/or composition, and the
discretion of the attending physician. The binding molecule,
recombinant receptor, cell and/or composition and/or the additional
agent and/or therapy can be administered to the patient at one
time, repeated or administered over a series of treatments.
[0720] C. Diagnostic and Detection Methods
[0721] Also provided are methods involving use of the provided
binding molecules, e.g., antibodies or antigen-binding fragments
thereof, in detection of ROR1, for example, in diagnostic and/or
prognostic methods in association with a ROR1-expressing disease or
condition. The methods in some embodiments include incubating a
biological sample with the antibody or antigen-binding fragment
thereof and/or administering the antibody or antigen-binding
fragment thereof to a subject. In certain embodiments, a biological
sample includes a cell or tissue, such as tumor or cancer tissue.
In certain embodiments, the contacting is under conditions
permissive for binding of the anti-ROR1 antibody to ROR1, and
detecting whether a complex is formed between the anti-ROR1
antibody and ROR1. Such a method may be an in vitro or in vivo
method. In one embodiment, an anti-ROR1 antibody (e.g.,
antigen-binding fragment) is used to select subjects eligible for
therapy with an anti-ROR1 antibody (e.g., antigen-binding fragment)
or recombinant receptor, e.g. where ROR1 is a biomarker for
selection of patients.
[0722] In some embodiments, a sample, such as a cell, tissue
sample, lysate, composition, or other sample derived therefrom is
contacted with the anti-ROR1 antibody (e.g., antigen-binding
fragment) and binding or formation of a complex between the
antibody and the sample (e.g., ROR1 in the sample) is determined or
detected. When binding in the test sample is demonstrated or
detected as compared to a reference cell of the same tissue type,
it may indicate the presence of an associated disease or condition.
In some embodiments, the sample is from human tissues.
[0723] Various known methods for detecting specific
antibody-antigen binding can be used. Exemplary immunoassays
include fluorescence polarization immunoassay (FPIA), fluorescence
immunoassay (FIA), enzyme immunoassay (EIA), nephelometric
inhibition immunoassay (NIA), enzyme linked immunosorbent assay
(ELISA), and radioimmunoassay (RIA). An indicator moiety, or label
group, can be attached to the subject antibodies and is selected so
as to meet the needs of various uses of the method which are often
dictated by the availability of assay equipment and compatible
immunoassay procedures. Exemplary labels include radionuclides
(e.g. .sup.125I, .sup.131I, .sup.35S, .sup.3H, or .sup.32P),
enzymes (e.g., alkaline phosphatase, horseradish peroxidase,
luciferase, or .beta.-galactosidase), fluorescent moieties or
proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or
BFP), or luminescent moieties (e.g., Qdot.TM. nanoparticles
supplied by the Quantum Dot Corporation, Palo Alto, Calif.).
General techniques to be used in performing the various
immunoassays noted above are known to those of ordinary skill in
the art.
[0724] For purposes of diagnosis, the antibodies (e.g.,
antigen-binding fragments) can be labeled with a detectable moiety
including but not limited to radioisotopes, fluorescent labels, and
various enzyme-substrate labels know in the art. Methods of
conjugating labels to an antibody are known.
[0725] In some embodiments, antibodies (e.g., antigen-binding
fragments) need not be labeled, and the presence thereof can be
detected using a labeled antibody which binds to the
antibodies.
[0726] The provided antibodies (e.g., antigen-binding fragments) in
some embodiments can be employed in any known assay method, such as
competitive binding assays, direct and indirect sandwich assays,
and immunoprecipitation assays. Zola, Monoclonal Antibodies: A
Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).
[0727] The antibodies (e.g., antigen-binding fragments) and
polypeptides can also be used for in vivo diagnostic assays, such
as in vivo imaging. Generally, the antibody is labeled with a
radionuclide (such as .sup.111In, .sup.99Tc, .sup.14C, .sup.131I,
.sup.125I, or .sup.3H) so that the cells or tissue of interest can
be localized in vivo following administration to a subject.
[0728] The antibody (e.g., antigen-binding fragment) may also be
used as staining reagent in pathology, e.g., using known
techniques.
V. ARTICLES OF MANUFACTURE OR KITS
[0729] Also provided are articles of manufacture or kit containing
the provided binding molecules (e.g., antibodies), recombinant
receptors (e.g., CARs), genetically engineered cells, and/or
compositions comprising the same. The articles of manufacture may
include a container and a label or package insert on or associated
with the container. Suitable containers include, for example,
bottles, vials, syringes, test tubes, IV solution bags, etc. The
containers may be formed from a variety of materials such as glass
or plastic. In some embodiments, the container has a sterile access
port. Exemplary containers include an intravenous solution bags,
vials, including those with stoppers pierceable by a needle for
injection. The article of manufacture or kit may further include a
package insert indicating that the compositions can be used to
treat a particular condition such as a condition described herein
(e.g., ROR1-expressing cancer). Alternatively, or additionally, the
article of manufacture or kit may further include another or the
same container comprising a pharmaceutically-acceptable buffer. It
may further include other materials such as other buffers,
diluents, filters, needles, and/or syringes.
[0730] The label or package insert may indicate that the
composition is used for treating the ROR1-expressing or
ROR1-associated disease, disorder or condition in an individual.
The label or a package insert, which is on or associated with the
container, may indicate directions for reconstitution and/or use of
the formulation. The label or package insert may further indicate
that the formulation is useful or intended for subcutaneous,
intravenous, or other modes of administration for treating or
preventing a ROR1-expressing or ROR1-associated disease, disorder
or condition in an individual. In some aspects, the label or
package insert can include instructions for use, for example
instructions for administering the antibody or antigen-binding
fragment thereof, the single chain cell surface protein, the
conjugate, the chimeric antigen receptor, the cell or the
composition, in some aspects in accord with any of the methods or
uses described herein.
[0731] The container in some embodiments holds a composition which
is by itself or combined with another composition effective for
treating, preventing and/or diagnosing the condition. The article
of manufacture or kit may include (a) a first container with a
composition contained therein (i.e., first medicament), wherein the
composition includes the antibody (e.g., anti-ROR1 antibody) or
antigen-binding fragment thereof or recombinant receptor (e.g.,
CAR); and (b) a second container with a composition contained
therein (i.e., second medicament), wherein the composition includes
a further agent, such as a cytotoxic or otherwise therapeutic
agent, and which article or kit further comprises instructions on
the label or package insert for treating the subject with the
second medicament, in an effective amount.
VI. DEFINITIONS
[0732] As used herein, reference to a "corresponding form" of an
antibody means that when comparing a property or activity of two
antibodies, the property is compared using the same form of the
antibody. For example, if it is stated that an antibody has greater
activity compared to the activity of the corresponding form of a
first antibody, that means that a particular form, such as an scFv
of that antibody, has greater activity compared to the scFv form of
the first antibody.
[0733] "Effector functions" refer to those biological activities
attributable to the Fc region of an antibody, which vary with the
antibody isotype. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors
(e.g. B cell receptor); and B cell activation.
[0734] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain that contains at least a
portion of the constant region. The term includes native sequence
Fc regions and variant Fc regions. In one embodiment, a human IgG
heavy chain Fc region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) of the Fc region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fc region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.,
1991.
[0735] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0736] An "isolated" antibody is one which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC). For
review of methods for assessment of antibody purity, see, e.g.,
Flatman et al., J. Chromatogr. B 848:79-87 (2007).
[0737] An "isolated" nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
[0738] "Isolated nucleic acid encoding an anti-ROR1 antibody"
refers to one or more nucleic acid molecules encoding antibody
heavy and light chains (or fragments thereof), including such
nucleic acid molecule(s) in a single vector or separate vectors,
and such nucleic acid molecule(s) present at one or more locations
in a host cell.
[0739] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
[0740] As used herein, "percent (%) amino acid sequence identity"
and "percent identity" and "sequence identity" when used with
respect to an amino acid sequence (reference polypeptide sequence)
is defined as the percentage of amino acid residues in a candidate
sequence (e.g., the subject antibody or fragment) that are
identical with the amino acid residues in the reference polypeptide
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity. Alignment for purposes of determining percent
amino acid sequence identity can be achieved in various ways that
are within the skill in the art, for instance, using publicly
available computer software such as BLAST, BLAST-2, ALIGN or
Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for aligning sequences, including any
algorithms needed to achieve maximal alignment over the full length
of the sequences being compared.
[0741] An amino acid substitution may include replacement of one
amino acid in a polypeptide with another amino acid. The
substitution may be a conservative amino acid substitution or a
non-conservative amino acid substitution. Amino acid substitutions
may be introduced into a binding molecule, e.g., antibody, of
interest and the products screened for a desired activity, e.g.,
retained/improved antigen binding, decreased immunogenicity, or
improved ADCC or CDC.
[0742] Amino acids generally can be grouped according to the
following common side-chain properties: [0743] (1) hydrophobic:
Norleucine, Met, Ala, Val, Leu, Ile; [0744] (2) neutral
hydrophilic: Cys, Ser, Thr, Asn, Gln; [0745] (3) acidic: Asp, Glu;
[0746] (4) basic: His, Lys, Arg; [0747] (5) residues that influence
chain orientation: Gly, Pro; [0748] (6) aromatic: Trp, Tyr,
Phe.
[0749] In some embodiments, conservative substitutions can involve
the exchange of a member of one of these classes for another member
of the same class. In some embodiments, non-conservative amino acid
substitutions can involve exchanging a member of one of these
classes for another class.
[0750] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors."
[0751] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0752] As used herein, the singular forms "a," "an," and "the"
include plural referents unless the context clearly dictates
otherwise. For example, "a" or "an" means "at least one" or "one or
more." It is understood that aspects, embodiments, and variations
described herein include "comprising," "consisting," and/or
"consisting essentially of" aspects, embodiments and
variations.
[0753] Throughout this disclosure, various aspects of the claimed
subject matter are presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the claimed subject matter.
Accordingly, the description of a range should be considered to
have specifically disclosed all the possible sub-ranges as well as
individual numerical values within that range. For example, where a
range of values is provided, it is understood that each intervening
value, between the upper and lower limit of that range and any
other stated or intervening value in that stated range is
encompassed within the claimed subject matter. The upper and lower
limits of these smaller ranges may independently be included in the
smaller ranges, and are also encompassed within the claimed subject
matter, subject to any specifically excluded limit in the stated
range. Where the stated range includes one or both of the limits,
ranges excluding either or both of those included limits are also
included in the claimed subject matter. This applies regardless of
the breadth of the range.
[0754] The term "about" as used herein refers to the usual error
range for the respective value readily known to the skilled person
in this technical field. Reference to "about" a value or parameter
herein includes (and describes) embodiments that are directed to
that value or parameter per se. For example, description referring
to "about X" includes description of "X".
[0755] As used herein, a "composition" refers to any mixture of two
or more products, substances, or compounds, including cells. It may
be a solution, a suspension, liquid, powder, a paste, aqueous,
non-aqueous or any combination thereof.
[0756] As used herein, a statement that a cell or population of
cells is "positive" for a particular marker refers to the
detectable presence on or in the cell of a particular marker,
typically a surface marker. When referring to a surface marker, the
term refers to the presence of surface expression as detected by
flow cytometry, for example, by staining with an antibody that
specifically binds to the marker and detecting said antibody,
wherein the staining is detectable by flow cytometry at a level
substantially above the staining detected carrying out the same
procedure with an isotype-matched control under otherwise identical
conditions and/or at a level substantially similar to that for cell
known to be positive for the marker, and/or at a level
substantially higher than that for a cell known to be negative for
the marker.
[0757] As used herein, a statement that a cell or population of
cells is "negative" for a particular marker refers to the absence
of substantial detectable presence on or in the cell of a
particular marker, typically a surface marker. When referring to a
surface marker, the term refers to the absence of surface
expression as detected by flow cytometry, for example, by staining
with an antibody that specifically binds to the marker and
detecting said antibody, wherein the staining is not detected by
flow cytometry at a level substantially above the staining detected
carrying out the same procedure with an isotype-matched control
under otherwise identical conditions, and/or at a level
substantially lower than that for cell known to be positive for the
marker, and/or at a level substantially similar as compared to that
for a cell known to be negative for the marker.
[0758] Unless defined otherwise, all terms of art, notations and
other technical and scientific terms or terminology used herein are
intended to have the same meaning as is commonly understood by one
of ordinary skill in the art to which the claimed subject matter
pertains. In some cases, terms with commonly understood meanings
are defined herein for clarity and/or for ready reference, and the
inclusion of such definitions herein should not necessarily be
construed to represent a substantial difference over what is
generally understood in the art.
VII. EXEMPLARY EMBODIMENTS
[0759] Among the provided embodiments are:
[0760] 1. An anti-ROR1 antibody or antigen-binding fragment
thereof, comprising: a heavy chain variable (V.sub.H) region, and a
light chain variable (V.sub.L) region, wherein the V.sub.H region
comprises a heavy chain complementarity determining region 1
(CDR-H1) comprising the sequence set forth in SEQ ID NO: 67, 82 or
52, a heavy chain complementarity determining region 2 (CDR-H2)
comprising the sequence set forth in SEQ ID NO: 71, 86, 56 or 97,
and a heavy chain complementarity determining region 3 (CDR-H3)
comprising the sequence set forth in SEQ ID NO: 73, 88, 58 or 99,
and the V.sub.L region comprises a light chain complementarity
determining region 1 (CDR-L1) comprising the sequence set forth in
SEQ ID NO: 75, 90 or 60, a light chain complementarity determining
region 2 (CDR-L2) comprising the sequence set forth in SEQ ID NO:
77, 92 or 62; and a light chain complementarity determining region
3 (CDR-L3) comprising the sequence set forth in SEQ ID NO: 79, 94
or 64.
[0761] 2. An anti-ROR1 antibody or antigen-binding fragment
thereof, comprising: a heavy chain variable (V.sub.H) region, and a
light chain variable (V.sub.L) region, wherein the V.sub.H region
comprises a heavy chain complementarity determining region 1
(CDR-H1), a heavy chain complementarity determining region 2
(CDR-H2) and a heavy chain complementarity determining region 3
(CDR-H3) comprising the sequence set forth in SEQ ID NOS:67, 71 and
73, respectively, and the V.sub.L region comprises a light chain
complementarity determining region 1 (CDR-L1), a light chain
complementarity determining region 2 (CDR-L2) and a light chain
complementarity determining region 3 (CDR-L3) comprising the
sequence set forth in SEQ ID NOS:75, 77 and 79, respectively;
[0762] the V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3
comprising the sequence set forth in SEQ ID NOS:82, 86 and 88,
respectively, and the V.sub.L region comprises a CDR-L1, a CDR-L2
and a CDR-L3 comprising the sequence set forth in SEQ ID NOS:90, 92
and 94, respectively;
[0763] the V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3
comprising the sequence set forth in SEQ ID NOS:52, 56 and 58,
respectively, and the V.sub.L region comprises a CDR-L1, a CDR-L2
and a CDR-L3 comprising the sequence set forth in SEQ ID NOS:60, 62
and 64, respectively; or
[0764] the V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3
comprising the sequence set forth in SEQ ID NOS:52, 97 and 99,
respectively, and the V.sub.L region comprises a CDR-L1, a CDR-L2
and a CDR-L3 comprising the sequence set forth in SEQ ID NOS:60, 62
and 64, respectively.
[0765] 3. The anti-ROR1 antibody or antigen-binding fragment
thereof of embodiment 1 or embodiment 2, wherein the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence
set forth in SEQ ID NOS:67, 71 and 73, respectively, and the
V.sub.L region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising
the sequence set forth in SEQ ID NOS:75, 77 and 79,
respectively.
[0766] 4. The anti-ROR1 antibody or antigen-binding fragment
thereof of embodiment 1 or embodiment 2, wherein the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence
set forth in SEQ ID NOS:82, 86 and 88, respectively, and the
V.sub.L region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising
the sequence set forth in SEQ ID NOS:90, 92 and 94,
respectively.
[0767] 5. An anti-ROR1 antibody or antigen-binding fragment
thereof, comprising: a heavy chain variable (V.sub.H) region and a
light chain variable (V.sub.L) region, wherein:
[0768] the V.sub.H region comprises a heavy chain complementarity
determining region 1 (CDR-H1), a heavy chain complementarity
determining region 2 (CDR-H2) and a heavy chain complementarity
determining region 3 (CDR-H3) contained within SEQ ID NO: 112, 121,
103 or 130, and the V.sub.L region comprises a light chain
complementarity determining region 1 (CDR-L1), a light chain
complementarity determining region 2 (CDR-L2) and a light chain
complementarity determining region 3 (CDR-L3) contained within SEQ
ID NO: 115, 124 or 106.
[0769] 6. An anti-ROR1 antibody or antigen-binding fragment
thereof, comprising: a heavy chain variable (V.sub.H) region and a
light chain variable (V.sub.L) region, wherein:
[0770] the V.sub.H region comprises a heavy chain complementarity
determining region 1 (CDR-H1), a heavy chain complementarity
determining region 2 (CDR-H2) and a heavy chain complementarity
determining region 3 (CDR-H3) contained within SEQ ID NO:112, and
the V.sub.L region comprises a light chain complementarity
determining region 1 (CDR-L1), a light chain complementarity
determining region 2 (CDR-L2) and a light chain complementarity
determining region 3 (CDR-L3) contained within SEQ ID NO:115;
[0771] the V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3
contained within SEQ ID NO:121, and the V.sub.L region comprises a
CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID: NO 124;
[0772] the V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3
contained within SEQ ID NO:103, and the V.sub.L region comprises a
CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID: NO 106;
or
[0773] the V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3
contained within SEQ ID NO:130, and the V.sub.L region comprises a
CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID: NO 106.
[0774] 7. The anti-ROR1 antibody or antigen-binding fragment
thereof of embodiment 5 or embodiment 6, wherein the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within SEQ ID
NO:112, and the V.sub.L region comprises a CDR-L1, a CDR-L2 and a
CDR-L3 contained within SEQ ID: NO 115.
[0775] 8. The anti-ROR1 antibody or antigen-binding fragment
thereof of embodiment 5 or embodiment 6, wherein the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within SEQ ID
NO:121, and the V.sub.L region comprises a CDR-L1, a CDR-L2 and a
CDR-L3 contained within SEQ ID: NO 124.
[0776] 9. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1, 2, 5 and 6, wherein:
[0777] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 112,
121, 103 or 130, and
[0778] the V.sub.L region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 115, 124
or 106.
[0779] 10. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1, 2, and 5-7, wherein:
[0780] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:112, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 115;
[0781] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:121, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:124;
[0782] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:103, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:106; or
[0783] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:130, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:106.
[0784] 11. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-10, wherein the V.sub.H region is
or comprises an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:112, and the V.sub.L region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 115.
[0785] 12. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-10, wherein the V.sub.H region is
or comprises an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:121, and the V.sub.L region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:124.
[0786] 13. An anti-ROR1 antibody or antigen-binding fragment
thereof, comprising: a heavy chain variable (V.sub.H) region and a
light chain variable (V.sub.L) region, wherein:
[0787] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 112,
121, 103 or 130, and
[0788] the V.sub.L region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 115, 124
or 106.
[0789] 14. An anti-ROR1 antibody or antigen-binding fragment
thereof, comprising: a heavy chain variable (V.sub.H) region and a
light chain variable (V.sub.L) region, wherein:
[0790] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:112, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 115;
[0791] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:121, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:124;
[0792] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:103, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:106; or
[0793] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:130, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:106.
[0794] 15. The anti-ROR1 antibody or antigen-binding fragment
thereof of embodiment 13 or embodiment 14, wherein the V.sub.H
region is or comprises an amino acid sequence having at least at or
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity to SEQ ID NO:112, and the V.sub.L region
is or comprises an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 115.
[0795] 16. The anti-ROR1 antibody or antigen-binding fragment
thereof of embodiment 13 or embodiment 14, wherein the V.sub.H
region is or comprises an amino acid sequence having at least at or
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity to SEQ ID NO:121, and the V.sub.L region
is or comprises an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:124.
[0796] 17. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1, 2, 5, 6, 9, 10, 13 and 14,
wherein:
[0797] the V.sub.H region is or comprises the sequence set forth in
SEQ ID NO: 112, 121, 103 or 130, and
[0798] the V.sub.L region is or comprises the sequence set forth in
SEQ ID NO: 115, 124 or 106.
[0799] 18. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1, 2, 5, 6, 9, 10, 13, 14 and 17,
wherein:
[0800] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 112 and 115,
respectively;
[0801] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 121 and 124,
respectively;
[0802] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 103 and 106, respectively;
or
[0803] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 130 and 106,
respectively.
[0804] 19. An anti-ROR1 antibody or antigen-binding fragment
thereof, comprising: a heavy chain variable (V.sub.H) region and a
light chain variable (V.sub.L) region, wherein:
[0805] the V.sub.H region is or comprises the sequence set forth in
SEQ ID NO: 112, 121, 103 or 130, and
[0806] the V.sub.L region is or comprises the sequence set forth in
SEQ ID NO: 115, 124 or 106.
[0807] 20. An anti-ROR1 antibody or antigen-binding fragment
thereof, comprising: a heavy chain variable (V.sub.H) region and a
light chain variable (V.sub.L) region, wherein:
[0808] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 112 and 115,
respectively;
[0809] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 121 and 124,
respectively;
[0810] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 103 and 106, respectively;
or
[0811] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 130 and 106,
respectively.
[0812] 21. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-20, wherein the V.sub.H region and
the V.sub.L region are or comprise the sequence set forth in SEQ ID
NOS: 112 and 115, respectively.
[0813] 22. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-20, wherein the V.sub.H region and
the V.sub.L region are or comprise the sequence set forth in SEQ ID
NOS:121 and 124, respectively.
[0814] 23. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1, 2, 5, 6, 9, 10, 13, 14 and 17-20,
wherein the V.sub.H region is or comprises the amino acid sequence
encoded by SEQ ID NO: 110, 119, 101 or 128 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 110, 119, 101 or 128, and the V.sub.L
region is or comprises the amino acid sequence encoded by SEQ ID
NO: 113, 122 or 104, or a nucleic acid sequence having at least at
or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 113, 122 or 104.
[0815] 24. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1, 2, 5, 6, 9, 10, 13, 14, 17-20 and
23, wherein the V.sub.H region is or comprises the amino acid
sequence encoded by SEQ ID NO: 111, 120, 102 or 129, and the
V.sub.L region is or comprises the amino acid sequence encoded by
SEQ ID NO: 114, 123, 105 or 131.
[0816] 25. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-24, wherein the V.sub.H region is
or comprises the amino acid sequence encoded by SEQ ID NO: 110 or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 110, and the V.sub.L
region is or comprises the amino acid sequence encoded by SEQ ID
NO: 113, or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 113.
[0817] 26. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-25, wherein the V.sub.H region is
or comprises the amino acid sequence encoded by SEQ ID NO: 111, and
the V.sub.L region is or comprises the amino acid sequence encoded
by SEQ ID NO: 114.
[0818] 27. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-24, wherein the V.sub.H region is
or comprises the amino acid sequence encoded by SEQ ID NO: 119 or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 119, and the V.sub.L
region is or comprises the amino acid sequence encoded by SEQ ID
NO: 122, or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 122.
[0819] 28. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-25, wherein the V.sub.H region is
or comprises the amino acid sequence encoded by SEQ ID NO: 120, and
the V.sub.L region is or comprises the amino acid sequence encoded
by SEQ ID NO: 123.
[0820] 29. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-28, wherein said anti-ROR1 antibody
or antigen-binding fragment thereof is isolated.
[0821] 30. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-29, wherein said anti-ROR1 antibody
or antigen-binding fragment thereof is recombinant.
[0822] 31. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-30, wherein at least a portion of
the V.sub.H region and the V.sub.L region is human or is from a
human protein.
[0823] 32. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-31, wherein the antigen-binding
fragment thereof comprises a single chain fragment, optionally a
single chain Fv (scFv).
[0824] 33. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-32, wherein the V.sub.H region is
amino-terminal to the V.sub.L region.
[0825] 34. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-32, wherein the V.sub.H region is
carboxy-terminal to the V.sub.L region.
[0826] 35. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-34, when the V.sub.H region and the
V.sub.L region are joined by a flexible linker.
[0827] 36. The anti-ROR1 antibody or antigen-binding fragment
thereof of embodiment 35, wherein the flexible linker comprises the
sequence set forth in SEQ ID NO:41.
[0828] 37. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 32, 33, 35 and 36, wherein the scFv
is or comprises the sequence set forth in SEQ ID NO: 118, 127, 109
or 134, or an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% sequence identity to SEQ ID NO: 118, 127, 109 or 134.
[0829] 38. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 32, 33 and 35-37, wherein the scFv is
or comprises the sequence set forth in SEQ ID NO: 118.
[0830] 39. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 32, 33 and 35-37, wherein the scFv is
or comprises the sequence set forth in SEQ ID NO: 127.
[0831] 40. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 32, 33 and 35-37, wherein the scFv is
or comprises the amino acid sequence encoded by SEQ ID NO: 116,
125, 107 or 132 or a nucleic acid sequence having at least at or
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:
116, 125, 107 or 132.
[0832] 41. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 32, 33, 35-37 and 40, wherein the
scFv is or comprises the amino acid sequence encoded by SEQ ID NO:
117, 126, 108 or 133.
[0833] 42. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 32-41, wherein the scFv is or
comprises the amino acid sequence encoded by SEQ ID NO: 116 or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 116.
[0834] 43. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 32-42, wherein the scFv is or
comprises the amino acid sequence encoded by SEQ ID NO: 117.
[0835] 44. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 32-41, wherein the scFv is or
comprises the amino acid sequence encoded by SEQ ID NO: 125 or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 125.
[0836] 45. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 32-41 and 43, wherein the scFv is or
comprises the amino acid sequence encoded by SEQ ID NO: 126.
[0837] 46. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-45, wherein the anti-ROR1 antibody
or fragment further comprises at least a portion of an
immunoglobulin constant region or a variant thereof.
[0838] 47. The anti-ROR1 antibody or antigen-binding fragment
thereof of embodiment 46, wherein the portion of an immunoglobulin
constant region comprises at least a portion of a hinge region or a
variant thereof.
[0839] 48. The anti-ROR1 antibody or antigen-binding fragment
thereof of embodiment 46 or embodiment 47, wherein the at least a
portion of an immunoglobulin constant region or a variant thereof
comprises at least a portion of a CH2 region and/or a CH3 region or
a variant thereof.
[0840] 49. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 46-48, wherein the at least a portion
of an immunoglobulin constant region or a variant thereof is human
or from a human protein or a variant thereof.
[0841] 50. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-49, wherein said anti-ROR1 antibody
or antigen-binding fragment thereof specifically binds to a
Receptor tyrosine kinase-like orphan receptor 1 (ROR1) protein,
optionally a human ROR1.
[0842] 51. The anti-ROR1 antibody or antigen-binding fragment
thereof of embodiment 50, wherein the human ROR1 protein comprises
an amino acid sequence set forth in SEQ ID NO: 144, 145 or 146.
[0843] 52. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-51, wherein said anti-ROR1 antibody
or antigen-binding fragment thereof does not bind to, is not
cross-reactive to, or binds at a lower level or degree or affinity
to a Receptor tyrosine kinase-like orphan receptor 2 (ROR2)
protein, optionally a human ROR2.
[0844] 53. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-52, wherein the extent, level or
degree or affinity of binding of said anti-ROR1 antibody or
antigen-binding fragment thereof to a human ROR2 is at least at or
about 75%, 80%, 90%, 95% or 99% less than the extent, level or
degree or affinity of binding to a human ROR1.
[0845] 54. A single chain cell-surface protein, comprising the
anti-ROR1 antibody or antigen-binding fragment thereof of any of
embodiments 1-53 and 244-265.
[0846] 55. A conjugate, comprising the anti-ROR1 antibody or
antigen-binding fragment thereof of any of embodiments 1-53 and
244-265 and a heterologous molecule or moiety.
[0847] 56. The conjugate of embodiment 55, wherein the heterologous
molecule or moiety is a therapeutic moiety.
[0848] 57. An anti-ROR1 chimeric antigen receptor (CAR) comprising
an extracellular antigen-binding domain comprising the anti-ROR1
antibody or antigen-binding fragment thereof of any of embodiments
1-53 and 244-265 and an intracellular signaling region.
[0849] 58. An anti-ROR1 chimeric antigen receptor (CAR)
comprising:
[0850] an extracellular antigen-binding domain comprising a heavy
chain variable (V.sub.H) region, and a light chain variable
(V.sub.L) region, and an intracellular signaling region, wherein
the V.sub.H region comprises a heavy chain complementarity
determining region 1 (CDR-H1) comprising the sequence set forth in
SEQ ID NO: 67, 82 or 52, a heavy chain complementarity determining
region 2 (CDR-H2) comprising the sequence set forth in SEQ ID NO:
71, 86, 56 or 97, and a heavy chain complementarity determining
region 3 (CDR-H3) comprising the sequence set forth in SEQ ID NO:
73, 88, 58 or 99, and the V.sub.L region comprises a light chain
complementarity determining region 1 (CDR-L1) comprising the
sequence set forth in SEQ ID NO: 75, 90 or 60, a light chain
complementarity determining region 2 (CDR-L2) comprising the
sequence set forth in SEQ ID NO: 77, 92 or 62; and a light chain
complementarity determining region 3 (CDR-L3) comprising the
sequence set forth in SEQ ID NO: 79, 94 or 64.
[0851] 59. An anti-ROR1 chimeric antigen receptor (CAR)
comprising:
[0852] an extracellular antigen-binding domain comprising a heavy
chain variable (V.sub.H) region, and a light chain variable
(V.sub.L) region, and an intracellular signaling region, wherein
the V.sub.H region comprises a heavy chain complementarity
determining region 1 (CDR-H1), a heavy chain complementarity
determining region 2 (CDR-H2) and a heavy chain complementarity
determining region 3 (CDR-H3) comprising the sequence set forth in
SEQ ID NOS:67, 71 and 73, respectively, and the V.sub.L region
comprises a light chain complementarity determining region 1
(CDR-L1), a light chain complementarity determining region 2
(CDR-L2) and a light chain complementarity determining region 3
(CDR-L3) comprising the sequence set forth in SEQ ID NOS:75, 77 and
79, respectively;
[0853] the V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3
comprising the sequence set forth in SEQ ID NOS:82, 86 and 88,
respectively, and the V.sub.L region comprises a CDR-L1, a CDR-L2
and a CDR-L3 comprising the sequence set forth in SEQ ID NOS:90, 92
and 94, respectively;
[0854] the V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3
comprising the sequence set forth in SEQ ID NOS:52, 56 and 58,
respectively, and the V.sub.L region comprises a CDR-L1, a CDR-L2
and a CDR-L3 comprising the sequence set forth in SEQ ID NOS:60, 62
and 64, respectively; or
[0855] the V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3
comprising the sequence set forth in SEQ ID NOS:52, 97 and 99,
respectively, and the V.sub.L region comprises a CDR-L1, a CDR-L2
and a CDR-L3 comprising the sequence set forth in SEQ ID NOS:60, 62
and 64, respectively.
[0856] 60. The anti-ROR1 chimeric antigen receptor of embodiment 58
or embodiment 59, wherein the V.sub.H region comprises a CDR-H1, a
CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID
NOS:67, 71 and 73, respectively, and the V.sub.L region comprises a
CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in
SEQ ID NOS:75, 77 and 79, respectively.
[0857] 61. The anti-ROR1 chimeric antigen receptor of embodiment 58
or embodiment 59, wherein the V.sub.H region comprises a CDR-H1, a
CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID
NOS:82, 86 and 88, respectively, and the V.sub.L region comprises a
CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in
SEQ ID NOS:90, 92 and 94, respectively.
[0858] 62. An anti-ROR1 chimeric antigen receptor (CAR)
comprising:
[0859] an extracellular antigen-binding domain comprising a heavy
chain variable (V.sub.H) region and a light chain variable
(V.sub.L) region, and an intracellular signaling region,
wherein:
[0860] the V.sub.H region comprises a heavy chain complementarity
determining region 1 (CDR-H1), a heavy chain complementarity
determining region 2 (CDR-H2) and a heavy chain complementarity
determining region 3 (CDR-H3) contained within SEQ ID NO: 112, 121,
103 or 130, and
[0861] the V.sub.L region comprises a light chain complementarity
determining region 1 (CDR-L1), a light chain complementarity
determining region 2 (CDR-L2) and a light chain complementarity
determining region 3 (CDR-L3) contained within SEQ ID NO: 115, 124
or 106.
[0862] 63. An anti-ROR1 chimeric antigen receptor (CAR)
comprising:
[0863] an extracellular antigen-binding domain comprising a heavy
chain variable (V.sub.H) region and a light chain variable
(V.sub.L) region, and an intracellular signaling region,
wherein:
[0864] the V.sub.H region comprises a heavy chain complementarity
determining region 1 (CDR-H1), a heavy chain complementarity
determining region 2 (CDR-H2) and a heavy chain complementarity
determining region 3 (CDR-H3) contained within SEQ ID NO:112, and
the V.sub.L region comprises a light chain complementarity
determining region 1 (CDR-L1), a light chain complementarity
determining region 2 (CDR-L2) and a light chain complementarity
determining region 3 (CDR-L3) contained within SEQ ID NO:115;
[0865] the V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3
contained within SEQ ID NO:121, and the V.sub.L region comprises a
CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID: NO 124;
[0866] the V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3
contained within SEQ ID NO:103, and the V.sub.L region comprises a
CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID: NO 106;
or
[0867] the V.sub.H region comprises a CDR-H1, a CDR-H2 and a CDR-H3
contained within SEQ ID NO:130, and the V.sub.L region comprises a
CDR-L1, a CDR-L2 and a CDR-L3 contained within SEQ ID: NO 106.
[0868] 64. The anti-ROR1 chimeric antigen receptor of embodiment 62
or embodiment 63, wherein the V.sub.H region comprises a CDR-H1, a
CDR-H2 and a CDR-H3 contained within SEQ ID NO:112, and the V.sub.L
region comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within
SEQ ID: NO 115.
[0869] 65. The anti-ROR1 chimeric antigen receptor of embodiment 62
or embodiment 63, wherein the V.sub.H region comprises a CDR-H1, a
CDR-H2 and a CDR-H3 contained within SEQ ID NO:121, and the V.sub.L
region comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within
SEQ ID: NO 124.
[0870] 66. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58, 59, 62 and 63, wherein:
[0871] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 112,
121, 103 or 130, and
[0872] the V.sub.L region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 115, 124
or 106.
[0873] 67. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58, 59, and 62-64, wherein:
[0874] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:112, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 115;
[0875] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:121, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:124;
[0876] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:103, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:106; or
[0877] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:130, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:106.
[0878] 68. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-67, wherein the V.sub.H region is or comprises an
amino acid sequence having at least at or about 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID NO:112, and the V.sub.L region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 115.
[0879] 69. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-67, wherein the V.sub.H region is or comprises an
amino acid sequence having at least at or about 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID NO:121, and the V.sub.L region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:124.
[0880] 70. An anti-ROR1 chimeric antigen receptor (CAR)
comprising:
[0881] an extracellular antigen-binding domain comprising a heavy
chain variable (V.sub.H) region and a light chain variable
(V.sub.L) region, and an intracellular signaling region,
wherein:
[0882] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 112,
121, 103 or 130, and
[0883] the V.sub.L region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 115, 124
or 106.
[0884] 71. An anti-ROR1 chimeric antigen receptor (CAR)
comprising:
[0885] an extracellular antigen-binding domain comprising a heavy
chain variable (V.sub.H) region and a light chain variable
(V.sub.L) region, and an intracellular signaling region,
wherein:
[0886] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:112, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 115;
[0887] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:121, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:124;
[0888] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:103, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:106; or
[0889] the V.sub.H region is or comprises an amino acid sequence
having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:130, and
the V.sub.L region is or comprises an amino acid sequence having at
least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:106.
[0890] 72. The anti-ROR1 chimeric antigen receptor of embodiment 70
or embodiment 71, wherein the V.sub.H region is or comprises an
amino acid sequence having at least at or about 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID NO:112, and the V.sub.L region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 115.
[0891] 73. The anti-ROR1 chimeric antigen receptor of embodiment 70
or embodiment 71, wherein the V.sub.H region is or comprises an
amino acid sequence having at least at or about 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID NO:121, and the V.sub.L region is or comprises an amino acid
sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO:124.
[0892] 74. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58, 59, 62, 63, 66, 67, 70 and 71, wherein:
[0893] the V.sub.H region is or comprises the sequence set forth in
SEQ ID NO: 112, 121, 103 or 130, and
[0894] the V.sub.L region is or comprises the sequence set forth in
SEQ ID NO: 115, 124 or 106.
[0895] 75. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58, 59, 62, 63, 66, 67, 70, 71 and 74, wherein:
[0896] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 112 and 115,
respectively;
[0897] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 121 and 124,
respectively;
[0898] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 103 and 106, respectively;
or
[0899] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 130 and 106,
respectively.
[0900] 76. An anti-ROR1 chimeric antigen receptor (CAR)
comprising:
[0901] an extracellular antigen-binding domain comprising a heavy
chain variable (V.sub.H) region and a light chain variable
(V.sub.L) region, and an intracellular signaling region,
wherein:
[0902] the V.sub.H region is or comprises the sequence set forth in
SEQ ID NO: 112, 121, 103 or 130, and
[0903] the V.sub.L region is or comprises the sequence set forth in
SEQ ID NO: 115, 124 or 106.
[0904] 77. An anti-ROR1 chimeric antigen receptor (CAR)
comprising:
[0905] an extracellular antigen-binding domain comprising a heavy
chain variable (V.sub.H) region and a light chain variable
(V.sub.L) region, and an intracellular signaling region,
wherein:
[0906] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 112 and 115,
respectively;
[0907] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 121 and 124,
respectively;
[0908] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 103 and 106, respectively;
or
[0909] the V.sub.H region and the V.sub.L region are or comprise
the sequence set forth in SEQ ID NOS: 130 and 106,
respectively.
[0910] 78. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-77, wherein the V.sub.H region and the V.sub.L
region are or comprise the sequence set forth in SEQ ID NOS: 112
and 115, respectively.
[0911] 79. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-77, wherein the V.sub.H region and the V.sub.L
region are or comprise the sequence set forth in SEQ ID NOS:121 and
124, respectively.
[0912] 80. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58, 59, 62, 63, 66, 67, 70, 71 and 74-77, wherein the
V.sub.H region is or comprises the amino acid sequence encoded by
SEQ ID NO: 110, 119, 101 or 128 or a nucleic acid sequence having
at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID NO: 110, 119, 101 or 128, and the V.sub.L region is or
comprises the amino acid sequence encoded by SEQ ID NO: 113, 122 or
104, or a nucleic acid sequence having at least at or about 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 113, 122 or
104.
[0913] 81. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58, 59, 62, 63, 66, 67, 70, 71, 74-77 and 80, wherein
the V.sub.H region is or comprises the amino acid sequence encoded
by SEQ ID NO: 111, 120, 102 or 129, and the V.sub.L region is or
comprises the amino acid sequence encoded by SEQ ID NO: 114, 123,
105 or 131.
[0914] 82. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-81, wherein the V.sub.H region is or comprises the
amino acid sequence encoded by SEQ ID NO: 110 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 110, and the V.sub.L region is or
comprises the amino acid sequence encoded by SEQ ID NO: 113, or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 113.
[0915] 83. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-82, wherein the V.sub.H region is or comprises the
amino acid sequence encoded by SEQ ID NO: 111, and the V.sub.L
region is or comprises the amino acid sequence encoded by SEQ ID
NO: 114.
[0916] 84. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-81, wherein the V.sub.H region is or comprises the
amino acid sequence encoded by SEQ ID NO: 119 or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 119, and the V.sub.L region is or
comprises the amino acid sequence encoded by SEQ ID NO: 122, or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 122.
[0917] 85. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-82, wherein the V.sub.H region is or comprises the
amino acid sequence encoded by SEQ ID NO: 120, and the V.sub.L
region is or comprises the amino acid sequence encoded by SEQ ID
NO: 123.
[0918] 86. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-85, wherein said anti-ROR1 antibody or
antigen-binding fragment thereof is isolated.
[0919] 87. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-86, wherein said anti-ROR1 antibody or
antigen-binding fragment thereof is recombinant.
[0920] 88. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-87, wherein at least a portion of the V.sub.H region
and the V.sub.L region is human or is from a human protein.
[0921] 89. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-88, wherein the antigen-binding fragment thereof
comprises a single chain fragment, optionally a single chain Fv
(scFv).
[0922] 90. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-89, wherein the V.sub.H region is amino-terminal to
the V.sub.L region.
[0923] 91. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-89, wherein the V.sub.H region is carboxy-terminal
to the V.sub.L region.
[0924] 92. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-91, when the V.sub.H region and the V.sub.L region
are joined by a flexible linker.
[0925] 93. The anti-ROR1 chimeric antigen receptor of embodiment
92, wherein the flexible linker comprises the sequence set forth in
SEQ ID NO:41.
[0926] 94. The anti-ROR1 chimeric antigen receptor of any of
embodiments 89, 90, 92 and 93, wherein the scFv is or comprises the
sequence set forth in SEQ ID NO: 118, 127, 109 or 134, or an amino
acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence
identity to SEQ ID NO: 118, 127, 109 or 134.
[0927] 95. The anti-ROR1 chimeric antigen receptor of any of
embodiments 89, 90 and 92-94, wherein the scFv is or comprises the
sequence set forth in SEQ ID NO: 118.
[0928] 96. The anti-ROR1 chimeric antigen receptor of any of
embodiments 89, 90 and 92-94, wherein the scFv is or comprises the
sequence set forth in SEQ ID NO: 127.
[0929] 97. The anti-ROR1 chimeric antigen receptor of any of
embodiments 89, 90 and 92-94, wherein the scFv is or comprises the
amino acid sequence encoded by SEQ ID NO: 116, 125, 107 or 132 or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 116, 125, 107 or
132.
[0930] 98. The anti-ROR1 chimeric antigen receptor of any of
embodiments 89, 90, 92-94 and 40, wherein the scFv is or comprises
the amino acid sequence encoded by SEQ ID NO: 117, 126, 108 or
133.
[0931] 99. The anti-ROR1 chimeric antigen receptor of any of
embodiments 89-98, wherein the scFv is or comprises the amino acid
sequence encoded by SEQ ID NO: 116 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 116.
[0932] 100. The anti-ROR1 chimeric antigen receptor of any of
embodiments 89-99, wherein the scFv is or comprises the amino acid
sequence encoded by SEQ ID NO: 117.
[0933] 101. The anti-ROR1 chimeric antigen receptor of any of
embodiments 89-98, wherein the scFv is or comprises the amino acid
sequence encoded by SEQ ID NO: 125 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 125.
[0934] 102. The anti-ROR1 chimeric antigen receptor of any of
embodiments 89-98 and 100, wherein the scFv is or comprises the
amino acid sequence encoded by SEQ ID NO: 126.
[0935] 103. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-102, further comprising a spacer.
[0936] 104. The anti-ROR1 chimeric antigen receptor of embodiment
103, wherein the spacer comprises at least a portion of an
immunoglobulin or a variant thereof.
[0937] 105. The anti-ROR1 chimeric antigen receptor of embodiment
103 or embodiment 104, wherein the spacer comprises at least a
portion of a hinge region of an immunoglobulin or a variant
thereof.
[0938] 106. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-105, wherein the spacer is less than at or about 15
amino acids in length.
[0939] 107. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-106, wherein the spacer is or comprises an amino
acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO:1, 26, 27, 29, 31, 32, 33 or 135.
[0940] 108. The anti-ROR1 chimeric antigen receptor of any of
embodiments 105-107, wherein the at least a portion of a hinge
region comprises all or a portion of an IgG4 hinge region,
optionally a human IgG4 hinge region, or a variant thereof.
[0941] 109. The anti-ROR1 chimeric antigen receptor of any of
embodiments 105-107, wherein the at least a portion of a hinge
region comprises all or a portion of an IgG2 hinge region,
optionally a human IgG2 hinge region, or a variant thereof.
[0942] 110. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-109, wherein the spacer is or comprises the
sequence set forth in SEQ ID NO: 135.
[0943] 111. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-110, wherein the spacer is or comprises the amino
acid sequence encoded by SEQ ID NO: 192 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 192.
[0944] 112. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-111, wherein the spacer is or comprises the amino
acid sequence encoded by SEQ ID NO: 136.
[0945] 113. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-112, wherein the spacer comprises at least a
portion of a CH3 region of an immunoglobulin or a variant
thereof.
[0946] 114. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-113, wherein the at least a portion of a CH3 region
comprises all or a portion of an IgG4 CH3 and/or an IgG2 CH3,
wherein the IgG4 CH3 is optionally a human IgG4 CH3 and the IgG2
CH3 is optionally a human IgG2 CH3.
[0947] 115. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-114, wherein the spacer comprises at least a
portion of a hinge region and at least a portion of a CH3 region of
an immunoglobulin or a variant thereof.
[0948] 116. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-115, wherein the spacer is at or about 111, 112,
113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 or 125
amino acids in length, or has a length between any of the
foregoing.
[0949] 117. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-116, wherein the spacer is at or about 120 amino
acids in length.
[0950] 118. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-117, wherein the spacer is or comprises an amino
acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO: 138.
[0951] 119. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-118, wherein the spacer is or comprises the
sequence set forth in SEQ ID NO:138.
[0952] 120. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-119, wherein the spacer is or comprises the amino
acid sequence encoded by SEQ ID NO: 193 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 193.
[0953] 121. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-120, wherein the spacer is or comprises the amino
acid sequence encoded by SEQ ID NO: 139.
[0954] 122. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-121, wherein the spacer comprises at least a
portion of a CH2 of an immunoglobulin or a variant thereof.
[0955] 123. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-122, wherein the at least a portion of a CH2 region
comprises all or a portion of an IgG4 CH2 and/or an IgG2 CH2,
wherein the IgG4 CH2 is optionally a human IgG4 CH2 and the IgG2
CH2 is optionally a human IgG2 CH2.
[0956] 124. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-123, wherein the spacer comprises at least a
portion of a hinge region, at least a portion of a CH2 and at least
a portion of a CH3 region of an immunoglobulin or a variant
thereof.
[0957] 125. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-124, wherein the spacer is at or about 130, 140,
150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229 or 230 amino acids in length, or has a length
between any of the foregoing.
[0958] 126. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-125, wherein one or more of the hinge region, the
CH2 region and the CH3 region comprises all or a portion of a CH2
region and all or a portion of a CH3 region from human IgG4.
[0959] 127. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-125, wherein one or more of the hinge region, the
CH2 region and the CH3 region is chimeric and comprises a hinge, a
CH2 region and a CH3 region from human IgG4 and human IgG2.
[0960] 128. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-125, wherein the spacer comprises a IgG4/2 chimeric
hinge region or a modified IgG4 hinge region comprising at least
one amino acid replacement compared to a human IgG4 hinge; an
IgG2/4 chimeric CH2 region; and an IgG4 CH3 region.
[0961] 129. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-125, wherein the spacer is or comprises an amino
acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO:194.
[0962] 130. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-129, wherein the spacer is or comprises the
sequence set forth in SEQ ID NO: 194.
[0963] 131. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-130, wherein the spacer is or comprises the amino
acid sequence encoded by SEQ ID NO: 195 or a nucleic acid sequence
having at least at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 195.
[0964] 132. The anti-ROR1 chimeric antigen receptor of any of
embodiments 103-131, wherein the spacer is or comprises the amino
acid sequence encoded by SEQ ID NO: 196.
[0965] 133. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-132, wherein the intracellular signaling region
comprises an intracellular signaling domain.
[0966] 134. The anti-ROR1 chimeric antigen receptor of embodiment
133, wherein the intracellular signaling domain is capable of
inducing a primary activation signal in a T cell, is a T cell
receptor (TCR) component and/or comprises an immunoreceptor
tyrosine-based activation motif (ITAM).
[0967] 135. The anti-ROR1 chimeric antigen receptor of embodiment
133 or embodiment 134, wherein the intracellular signaling domain
is or comprises a cytoplasmic signaling domain of a CD3-zeta
(CD3.zeta.) chain or a functional variant or signaling portion
thereof.
[0968] 136. The anti-ROR1 chimeric antigen receptor of any of
embodiments 133-135, wherein the intracellular signaling domain is
human or is from a human protein.
[0969] 137. The anti-ROR1 chimeric antigen receptor of any of
embodiments 133-136, wherein the intracellular signaling domain is
or comprises the sequence set forth in SEQ ID NO:13, 14 or 15, or
an amino acid sequence having at least at or about 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID
NO:13, 14 or 15.
[0970] 138. The anti-ROR1 chimeric antigen receptor of any of
embodiments 133-137, wherein the intracellular signaling domain is
or comprises the sequence set forth in SEQ ID NO:13.
[0971] 139. The anti-ROR1 chimeric antigen receptor of any of
embodiments 133-138, wherein the intracellular signaling region
further comprises a costimulatory signaling region.
[0972] 140. The anti-ROR1 chimeric antigen receptor of embodiment
139, wherein the costimulatory signaling region comprises an
intracellular signaling domain of a T cell costimulatory molecule
or a signaling portion thereof.
[0973] 141. The anti-ROR1 chimeric antigen receptor of embodiment
139 or embodiment 140, wherein the costimulatory signaling region
comprises an intracellular signaling domain of CD28, 4-1BB, or
ICOS, or a signaling portion thereof.
[0974] 142. The anti-ROR1 chimeric antigen receptor of any of
embodiments 140-141, wherein the costimulatory signaling region is
human or is from a human protein.
[0975] 143. The anti-ROR1 chimeric antigen receptor of any of
embodiments 140-142, wherein the costimulatory signaling region
comprises an intracellular signaling domain of CD28.
[0976] 144. The anti-ROR1 chimeric antigen receptor of any of
embodiments 140-143, wherein the costimulatory signaling region is
or comprises the sequence set forth in SEQ ID NO:10 or an amino
acid sequence having at least at or about 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:10.
[0977] 145. The anti-ROR1 chimeric antigen receptor of any of
embodiments 140-144, wherein the costimulatory signaling region
comprises an intracellular signaling domain of 4-1BB.
[0978] 146. The anti-ROR1 chimeric antigen receptor of any of
embodiments 140-142 and 145, wherein the costimulatory signaling
region is or comprises the sequence set forth in SEQ ID NO:12 or an
amino acid sequence having at least at or about 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:
12.
[0979] 147. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-146, further comprising a transmembrane region.
[0980] 148. The anti-ROR1 chimeric antigen receptor of any of
embodiments 140-147, wherein the costimulatory signaling region is
between the transmembrane region and the intracellular signaling
domain
[0981] 149. The anti-ROR1 chimeric antigen receptor of embodiment
147 or embodiment 148, wherein the transmembrane region is or
comprises a transmembrane domain from CD4, CD28, or CD8.
[0982] 150. The anti-ROR1 chimeric antigen receptor of any of
embodiments 147-149, wherein the transmembrane region is or
comprises a transmembrane domain derived from CD28.
[0983] 151. The anti-ROR1 chimeric antigen receptor of any of
embodiments 147-150, wherein the transmembrane region is human or
is from a human protein.
[0984] 152. The anti-ROR1 chimeric antigen receptor of any of
embodiments 147-151, wherein the transmembrane domain is or
comprises SEQ ID NO: 8 or an amino acid sequence having at least at
or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
sequence identity to SEQ ID NO: 8.
[0985] 153. The anti-ROR1 chimeric antigen receptor of any of
embodiments 147-152, wherein the encoded chimeric antigen receptor
comprises from its N to C terminus in order: the extracellular
antigen-binding domain, the spacer, the transmembrane region and
the intracellular signaling region.
[0986] 154. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-153, wherein the encoded chimeric antigen receptor
comprises, from its N to C terminus in order: an extracellular
antigen-binding domain comprising an scFv, a spacer comprising a
modified IgG4 hinge, optionally comprising the sequence set forth
in SEQ ID NO:135; a transmembrane domain, optionally a
transmembrane domain from a human CD28; and an intracellular
signaling region comprising a cytoplasmic signaling domain of a
CD3-zeta (CD3.zeta.) chain and an intracellular signaling domain of
a costimulatory signaling region, optionally comprising an
intracellular signaling domain of 4-1BB.
[0987] 155. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-153, wherein the encoded chimeric antigen receptor
comprises, from its N to C terminus in order: an extracellular
antigen-binding domain comprising an scFv, a spacer comprising a
modified IgG4 hinge-CH3, optionally comprising the sequence set
forth in SEQ ID NO: 138; a transmembrane domain, optionally a
transmembrane domain from a human CD28; and an intracellular
signaling region comprising a cytoplasmic signaling domain of a
CD3-zeta (CD3.zeta.) chain and an intracellular signaling domain of
a costimulatory signaling region, optionally comprising an
intracellular signaling domain of 4-1BB.
[0988] 156. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-155, wherein the anti-ROR1 chimeric antigen receptor
is or comprises the sequence set forth in SEQ ID NO: 184, 185, 186,
187, 188 or 189 or a sequence that exhibits at least 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the sequence set forth in SEQ ID NO: 184, 185,
186, 187, 188 or 189.
[0989] 157. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-156, wherein the anti-ROR1 chimeric antigen receptor
is or comprises the sequence set forth in SEQ ID NO: 184 or a
sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
sequence set forth in SEQ ID NO: 184.
[0990] 158. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-157, wherein the anti-ROR1 chimeric antigen receptor
is or comprises the sequence set forth in SEQ ID NO: 184.
[0991] 159. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-156, wherein the anti-ROR1 chimeric antigen receptor
is or comprises the sequence set forth in SEQ ID NO: 185 or a
sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
sequence set forth in SEQ ID NO:185.
[0992] 160. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-156 and 159, wherein the anti-ROR1 chimeric antigen
receptor is or comprises the sequence set forth in SEQ ID NO:
185.
[0993] 161. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-156, wherein the anti-ROR1 chimeric antigen receptor
is encoded by the sequence set forth in SEQ ID NO: 156, 157, 158,
159, 160 or 161 or a sequence that exhibits at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% sequence identity to the sequence set forth
in SEQ ID NO: 156, 157, 158, 159, 160 or 161.
[0994] 162. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-158 and 161, wherein the anti-ROR1 chimeric antigen
receptor is encoded by the sequence set forth in SEQ ID NO: 156 or
a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% sequence identity to the sequence set forth in SEQ ID NO:
156.
[0995] 163. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-158, 161 and 162, wherein the anti-ROR1 chimeric
antigen receptor is encoded by the sequence set forth in SEQ ID NO:
156.
[0996] 164. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-156 and 159-161, wherein the anti-ROR1 chimeric
antigen receptor is encoded by the sequence set forth in SEQ ID NO:
157 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the sequence set forth in SEQ ID
NO: 157.
[0997] 165. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-156, 159-161 and 164, wherein the anti-ROR1 chimeric
antigen receptor is encoded by the sequence set forth in SEQ ID NO:
157.
[0998] 166 The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-165, wherein said anti-ROR1 chimeric antigen
receptor specifically binds to a receptor tyrosine kinase-like
orphan receptor 1 (ROR1) protein, optionally a human ROR1.
[0999] 167. The anti-ROR1 chimeric antigen receptor of embodiment
166, wherein the human ROR1 protein comprises an amino acid
sequence set forth in SEQ ID NO: 144, 145 or 146.
[1000] 168. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-167, wherein said anti-ROR1 chimeric antigen
receptor does not bind to, is not cross-reactive to, or binds at a
lower level or degree or affinity to a receptor tyrosine
kinase-like orphan receptor 2 (ROR2) protein, optionally a human
ROR2.
[1001] 169. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-168, wherein the extent, level or degree or affinity
of binding of said anti-ROR1 chimeric antigen receptor to a human
ROR2 is at least at or about 75%, 80%, 90%, 95% or 99% less than
the extent, level or degree or affinity of binding to a human ROR1,
optionally under the same or substantially the same conditions or
assay.
[1002] 170. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-169, wherein said anti-ROR1 chimeric antigen
receptor exhibits the same, substantially the same or lower level
or degree of signaling or activity in the presence of a ROR2
protein, optionally a human ROR2, compared to the level or degree
of signaling or activity in the presence of a ROR1 protein,
optionally a human ROR1, optionally under the same or substantially
the same conditions or assay.
[1003] 171. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-170, wherein said anti-ROR1 chimeric antigen
receptor exhibits a level or degree of signaling or activity in the
presence of a human ROR2 that is at least at or about 75%, 80%,
90%, 95% or 99% less than the level or degree of signaling or
activity in the presence of a human ROR1, optionally under the same
or substantially the same conditions or assay.
[1004] 172. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-171, wherein said anti-ROR1 chimeric antigen
receptor exhibits the same, substantially the same or lower level
or degree of signaling or activity in the presence of a ROR2
protein, optionally a human ROR2, compared to a reference
ROR1-specific chimeric antigen receptor, optionally under the same
or substantially the same conditions or assay.
[1005] 173. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-172, wherein said anti-ROR1 chimeric antigen
receptor exhibits the same, substantially the same or lower level
or degree of signaling or activity in the presence of a ROR2
protein, optionally a human ROR2, compared to a reference
ROR1-specific chimeric antigen receptor, optionally under the same
or substantially the same conditions or assay.
[1006] 174. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-173, wherein said anti-ROR1 chimeric antigen
receptor exhibits the same, substantially the same or higher
antigen-specific signaling and/or antigen dependent activity or
signaling compared to a reference ROR1-specific chimeric antigen
receptor, optionally under the same or substantially the same
conditions or assay.
[1007] 175. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-174, wherein said anti-ROR1 chimeric antigen
receptor exhibits the same, substantially the same or lower tonic
signaling and/or antigen independent activity or signaling compared
to a reference ROR1-specific chimeric antigen receptor, optionally
under the same or substantially the same conditions or assay.
[1008] 176. The anti-ROR1 chimeric antigen receptor of any of
embodiments 58-175, wherein said anti-ROR1 chimeric antigen
receptor exhibits a level or degree of tonic signaling and/or
antigen independent activity or signaling that is at least at or
about 75%, 80%, 90%, 95% or 99% less than the level or degree of
tonic signaling and/or antigen independent activity of a reference
ROR1-specific chimeric antigen receptor, optionally under the same
or substantially the same conditions or assay.
[1009] 177. The anti-ROR1 chimeric antigen receptor of any of
embodiments 172-176, wherein the reference ROR1-specific chimeric
antigen receptor comprises the anti-ROR1 antibody R12 or the
anti-ROR1 antibody 2A2 or an antigen-binding fragment thereof,
optionally an scFv from R12 or 2A2.
[1010] 178. A polynucleotide comprising a nucleic acid encoding the
anti-ROR1 antibody or antigen-binding domain thereof of any of
embodiments 1-53 and 244-265, the single chain cell surface protein
of embodiment 54, the conjugate of embodiment 55 or embodiment 56
or the anti-ROR1 chimeric antigen receptor of any of embodiments
57-177 and 266-297.
[1011] 179. The polynucleotide of embodiment 178, wherein said
polynucleotide comprises a nucleic acid encoding the V.sub.H
comprising the sequence set forth in SEQ ID NO: 110, 119, 101 or
128 or a nucleic acid sequence having at least at or about 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 110, 119, 101
or 128, and a nucleic acid encoding the V.sub.L comprising the
sequence set forth in SEQ ID NO: 113, 122 or 104, or a nucleic acid
sequence having at least at or about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO: 113, 122 or 104.
[1012] 180. The polynucleotide of embodiment 178 or embodiment 179,
wherein said polynucleotide comprises a nucleic acid encoding the
V.sub.H comprising the sequence set forth in SEQ ID NO: 110 or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 110, and a nucleic acid
encoding the V.sub.L comprising the sequence set forth in SEQ ID
NO: 113, or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 113.
[1013] 181. The polynucleotide of embodiment 178 or embodiment 179,
wherein said polynucleotide comprises a nucleic acid encoding the
V.sub.H comprising the sequence set forth in SEQ ID NO: 119 or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 119, and a nucleic acid
encoding the V.sub.L comprising the sequence set forth in SEQ ID
NO: 122, or a nucleic acid sequence having at least at or about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 122.
[1014] 182. The polynucleotide of any of embodiments 178-180,
wherein said polynucleotide comprises a nucleic acid encoding the
scFv comprising the sequence set forth in SEQ ID NO: 116, 125, 107
or 132 or a nucleic acid sequence having at least at or about 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 116, 125, 107
or 132.
[1015] 183. The polynucleotide of any of embodiments 178-180 and
182, wherein said polynucleotide comprises a nucleic acid encoding
the scFv comprising the sequence set forth in SEQ ID NO: 116 or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 116.
[1016] 184. The polynucleotide of any of embodiments 178-180 and
182, wherein said polynucleotide comprises a nucleic acid encoding
the scFv comprising the sequence set forth in SEQ ID NO: 125 or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 125.
[1017] 185. The polynucleotide of any of embodiments 178-184,
wherein said polynucleotide comprises a nucleic acid encoding the
spacer comprising the sequence set forth in SEQ ID NO: 192 or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 192.
[1018] 186. The polynucleotide of any of embodiments 178-184,
wherein said polynucleotide comprises a nucleic acid encoding the
spacer comprising the sequence set forth in SEQ ID NO: 193 or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 193.
[1019] 187. The polynucleotide of any of embodiments 178-184,
wherein said polynucleotide comprises a nucleic acid encoding the
spacer comprising the sequence set forth in SEQ ID NO: 195 or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO: 195.
[1020] 188. The polynucleotide of embodiment 187, wherein the
nucleic acid encoding the anti-ROR1 antibody or antigen-binding
domain thereof, the single chain cell surface protein, the
conjugate or the anti-ROR1 chimeric antigen receptor comprises at
least one modified splice donor and/or splice acceptor site, said
modified splice donor and/or acceptor site comprising one or more
nucleotide modifications corresponding to a reference splice donor
site and/or reference splice acceptor site.
[1021] 189. The polynucleotide of embodiment 188, wherein the one
or more nucleotide modifications comprise a nucleic acid
substitution.
[1022] 190. The polynucleotide of embodiment 188 or embodiment 189,
wherein the reference splice donor and/or reference splice acceptor
sites are canonical, non-canonical, or cryptic splice sites.
[1023] 191. The polynucleotide of any of embodiment 188-190,
wherein:
[1024] the reference splice donor and/or reference splice acceptor
site(s) has a splice site prediction score of at least at or about
0.4, 0.5, 0.6, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.99 or 1.0;
and/or
[1025] the reference splice donor and/or reference splice acceptor
site(s) is/are predicted to be involved in a splice event with a
probability of at least at or about 40%, 50%, 60%, 70%, 75%, 80%,
85%, 90%, 95%, 99% or 100%.
[1026] 192. The polynucleotide of any of embodiment 188-191,
wherein:
[1027] the reference splice donor and/or reference splice acceptor
site(s) has a splice site prediction score of at least at or about
0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.99 or 1.0; and/or
[1028] the reference splice donor and/or reference splice acceptor
site(s) is/are predicted to be involved in a splice event with a
probability of at least at or about 70%, 75%, 80%, 85%, 90%, 95%,
99% or 100%.
[1029] 193. The polynucleotide of any of embodiments 188-192,
wherein at least one of the one or more nucleotide modifications
are within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues of the splice
site junction of the reference splice acceptor and/or reference
splice donor site.
[1030] 194. The polynucleotide of any of embodiments 188-193,
wherein the one or more nucleotide modifications is silent and/or
results in a degenerate codon and/or does not change the amino acid
sequence of the encoded protein.
[1031] 195. The polynucleotide of any of embodiments 188-194,
wherein upon expression of the polynucleotide in a cell, the
transcribed RNA, optionally messenger RNA (mRNA), from the
polynucleotide, exhibits at least at or about 70%, at or about 75%,
at or about 80%, at or about 85%, at or about 90%, or at or about
95% RNA homogeneity.
[1032] 196. The polynucleotide of any of embodiments 188-195,
wherein, upon expression in a cell, the transcribed RNA, optionally
messenger RNA (mRNA), from the polynucleotide exhibits reduced
heterogeneity compared to the heterogeneity of the mRNA transcribed
from a reference polynucleotide, said reference polynucleotide
encoding the same amino acid sequence as the polynucleotide,
wherein the reference polynucleotide differs by the presence of one
or more splice donor site and/or one or more splice acceptor site
in the nucleic acid encoding the spacer and/or comprises one or
more nucleotide modifications compared to the polynucleotide.
[1033] 197. The polynucleotide of embodiment 196, wherein the RNA
heterogeneity is reduced by greater than at or about 10%, 15%, 20%,
25%, 30%, 40% or 50% or more.
[1034] 198. The polynucleotide of embodiment 196 or embodiment 197,
wherein the transcribed RNA, optionally messenger RNA (mRNA), from
the reference polynucleotide exhibits greater than at or about 10%,
15%, 20%, 25%, 30%, 40% or 50% or more RNA heterogeneity.
[1035] 199. The polynucleotide of any of embodiments 188-198,
wherein the RNA homogeneity and/or heterogeneity is determined by
agarose gel electrophoresis, chip-based capillary electrophoresis,
analytical ultracentrifugation, field flow fractionation, or liquid
chromatography.
[1036] 200. The polynucleotide of any of embodiments 188-199,
wherein the polynucleotide is codon-optimized for expression in a
human cell.
[1037] 201. The polynucleotide of any of embodiments 178, 179, 182,
185, 186 and 188-200, wherein said polynucleotide comprises a
nucleic acid encoding the V.sub.H comprising the sequence set forth
in SEQ ID NO: 111, 120, 102 or 129, and a nucleic acid encoding the
V.sub.L comprising the sequence set forth in SEQ ID NO: 114, 123,
105 or 131.
[1038] 202. The polynucleotide of any of embodiments 178-180, 182,
183, 185, 186 and 198-201, wherein said polynucleotide comprises a
nucleic acid encoding the V.sub.H comprising the sequence set forth
in SEQ ID NO: 111, and a nucleic acid encoding the V.sub.L
comprising the sequence set forth in SEQ ID NO: 114.
[1039] 203. The polynucleotide of any of embodiments 178, 179, 181,
182, 184-186 and 198-201, wherein said polynucleotide comprises a
nucleic acid encoding the V.sub.H comprising the sequence set forth
in SEQ ID NO: 120, and a nucleic acid encoding the V.sub.L
comprising the sequence set forth in SEQ ID NO: 123.
[1040] 204. The polynucleotide of any of embodiments 178, 179, 182,
185, 186 and 188-201, wherein said polynucleotide comprises a
nucleic acid encoding the scFv comprising the sequence set forth in
SEQ ID NO: 117, 126, 108 or 133.
[1041] 205. The polynucleotide of any of embodiments 178-180, 182,
183, 185, 186, 198-201 and 204, wherein said polynucleotide
comprises a nucleic acid encoding the scFv comprising the sequence
set forth in SEQ ID NO: 117.
[1042] 206. The polynucleotide of any of embodiments 178, 179, 181,
182, 184-186, 198-201 and 204, wherein said polynucleotide
comprises a nucleic acid encoding the scFv comprising the sequence
set forth in SEQ ID NO: 126.
[1043] 207. The polynucleotide of any of embodiments 178-185 and
188-206, wherein said polynucleotide comprises a nucleic acid
encoding the spacer comprising the sequence set forth in SEQ ID
NO:136.
[1044] 208. The polynucleotide of any of embodiments 178-184, 186
and 188-206 wherein said polynucleotide comprises a nucleic acid
encoding the spacer comprising the sequence set forth in SEQ ID
NO:139.
[1045] 209. The polynucleotide of any of embodiments 178-184 and
187-206, wherein said polynucleotide comprises a nucleic acid
encoding the spacer comprising the sequence set forth in SEQ ID
NO:196.
[1046] 210. The polynucleotide of any of embodiments 178-186 and
188-209, wherein said polynucleotide comprises the sequence set
forth in SEQ ID NO: 156, 157, 158, 159, 160 or 161 or a sequence
that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the sequence set forth in SEQ ID NO: 156, 157, 158,
159, 160 or 161.
[1047] 211. The polynucleotide of any of embodiments 178-186 and
188-210, wherein said polynucleotide comprises the sequence set
forth in SEQ ID NO: 156 or a sequence that exhibits at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence
set forth in SEQ ID NO: 156.
[1048] 212. The polynucleotide of any of embodiments 178-186 and
188-211, wherein said polynucleotide comprises the sequence set
forth in SEQ ID NO: 156.
[1049] 213. The polynucleotide of any of embodiments 178-186 and
188-210, wherein said polynucleotide comprises the sequence set
forth in SEQ ID NO: 157 or a sequence that exhibits at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence
set forth in SEQ ID NO: 157.
[1050] 214. The polynucleotide of any of embodiments 178-186,
188-210 and 213, wherein said polynucleotide comprises the sequence
set forth in SEQ ID NO: 157.
[1051] 215. The polynucleotide of any of embodiments 178-214,
further comprising a CD33 signal sequence, a GM-CSF signal
sequence, a CD8 signal sequence or an Ig kappa signal sequence.
[1052] 216. The polynucleotide of any of embodiments 178-214,
further comprising a CD33 signal sequence.
[1053] 217. The polynucleotide of embodiment 215 or embodiment 216,
wherein the CD33 signal sequence is set forth in SEQ ID NO:190 or a
nucleic acid sequence having at least at or about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NO:190.
[1054] 218. A vector, comprising the polynucleotide of any of
embodiments 178-217.
[1055] 219. The vector of embodiment 218, wherein the vector is a
viral vector.
[1056] 220. The vector of embodiment 219, wherein the viral vector
is a retroviral vector or a lentiviral vector.
[1057] 221. A cell comprising the anti-ROR1 antibody or
antigen-binding fragment thereof of any of embodiments 1-53 and
244-265, the conjugate of embodiment 55 or embodiment 56, the
single chain cell surface protein of embodiment 54, the anti-ROR1
chimeric antigen receptor of any of embodiments 57-177 and 266-297,
the polynucleotide of any of embodiments 178-217, or the vector of
any of 218-220.
[1058] 222. The cell of embodiment 221, that is a lymphocyte.
[1059] 223. The cell of embodiment 222, that is an NK cell or a T
cell.
[1060] 224. The cell of embodiment 222 or embodiment 223, wherein
the cell is a T cell and the T cell is a CD4+ or a CD8+ T cell.
[1061] 225. The cell of any of embodiments 221-224, wherein the
cell is a primary cell obtained from a subject.
[1062] 226. The cell of any of embodiments 221-225, wherein, among
a plurality of the cells, less than at or about 10%, at or about
9%, at or about 8%, at or about 7%, at or about 5%, at or about 4%,
at or about 3%, at or about 2% or at or about 1% of the cells in
the plurality comprise an anti-ROR1 chimeric antigen receptor that
exhibits tonic signaling and/or antigen independent activity or
signaling.
[1063] 227. A composition comprising the anti-ROR1 antibody or
antigen-binding fragment thereof of any of embodiments 1-53 and
244-265, the conjugate of embodiment 55 or embodiment 56, the
single chain cell surface protein of embodiment 54 or the anti-ROR1
chimeric antigen receptor of any of embodiments 57-177 and
266-297.
[1064] 228. A composition comprising the cell of any of embodiments
221-226.
[1065] 229. The composition of embodiment 227 or embodiment 228,
further comprising a pharmaceutically acceptable excipient.
[1066] 230. The composition of embodiment 228 or embodiment 229,
wherein the composition comprises CD4+ and CD8+ T cells and the
ratio of CD4+ to CD8+ T cells is from at or about 1:3 to 3:1,
optionally at or about 1:2 to 2:1, optionally at or about 1:1.
[1067] 231. The composition of any of embodiments 227-230, wherein,
among a plurality of the cells, less than at or about 10%, at or
about 9%, at or about 8%, at or about 7%, at or about 5%, at or
about 4%, at or about 3%, at or about 2% or at or about 1% of the
cells in the plurality comprise an anti-ROR1 chimeric antigen
receptor that exhibits tonic signaling and/or antigen independent
activity or signaling.
[1068] 232. A method of treatment, comprising administering the
composition of any of embodiments 227-231 to a subject having a
disease or disorder associated with ROR1.
[1069] 233. A method of treatment, comprising administering the
anti-ROR1 antibody or antigen-binding fragment thereof of any of
embodiments 1-53 and 244-265, the conjugate of embodiment 55 or
embodiment 56, the single chain cell surface protein of embodiment
54, the anti-ROR1 chimeric antigen receptor of any of embodiments
57-177 and 266-297, the polynucleotide of any of embodiments
178-217, the vector of any of 218-220 or the cell of any of
embodiments 221-226 to a subject having a disease or disorder
associated with ROR1.
[1070] 234. A composition of any of embodiments 227-231 for use in
treating a disease or disorder associated with ROR1.
[1071] 235. Use of a composition of any of embodiments 227-231 for
the manufacture of a medicament for treating a disease or disorder
associated with ROR1.
[1072] 236. Use of a composition of any of embodiments 227-231 for
the treatment of a disease or disorder associated with ROR1.
[1073] 237. The method, the composition for use or the use of any
of embodiments 232-236 and 298-311, wherein the disease or disorder
associated with ROR1 is associated with ROR1 expression.
[1074] 238. The method, the composition for use or the use of any
of embodiments 232-237 and 298-311, wherein the disease or disorder
associated with ROR1 is a B cell-related disorder.
[1075] 239. The method, the composition for use or the use of any
of embodiments 232-238 and 298-311, wherein the disease or disorder
associated with ROR1 is a cancer.
[1076] 240. The method, the composition for use or the use of
embodiment 239, wherein the cancer is a ROR1-expressing cancer.
[1077] 241. The method, the composition for use or the use of
embodiment 240, wherein the ROR1-expressing cancer is selected from
the group consisting of B cell leukemia, lymphoma, B cell chronic
lymphocytic leukemia (CLL), acute myeloid leukemia (AML), acute
lymphocytic leukemia (ALL), Burkitt's Lymphoma, mantle cell
lymphoma (MCL), non-small cell lung cancer (NSCLC), neuroblastoma,
renal cell carcinoma, colon cancer, colorectal cancer, breast
cancer, epithelial squamous cell cancer, melanoma, myeloma, stomach
cancer, brain cancer, lung cancer, pancreatic cancer, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, prostate
cancer, testicular cancer, thyroid cancer, uterine cancer, adrenal
cancer and head and neck cancer.
[1078] 242. A kit comprising the anti-ROR1 antibody or
antigen-binding fragment thereof of any of embodiments 1-53 and
244-265, the single chain cell surface protein of embodiment 54,
the conjugate of embodiment 55 or embodiment 56, the anti-ROR1
chimeric antigen receptor of any of embodiments 57-177 and 266-297,
the cell of embodiment 221-226 or the composition of any of
embodiments 227-231, and instructions for use, optionally wherein
the instructions are for administering the anti-ROR1 antibody or
antigen-binding fragment thereof, the single chain cell surface
protein, the conjugate, the anti-ROR1 chimeric antigen receptor,
the cell or the composition, optionally in accord with the method,
the composition for use or the use of any of embodiments 234-241
and 298-311.
[1079] 243. An article of manufacture comprising the anti-ROR1
antibody or antigen-binding fragment thereof of any of embodiments
1-53 and 244-265, the single chain cell surface protein of
embodiment 54, the conjugate of embodiment 55 or embodiment 56, the
anti-ROR1 chimeric antigen receptor of any of embodiments 57-177
and 266-297, the cell of embodiment 221-226, the composition of any
of embodiments 227-231 or the kit of embodiment 242.
[1080] 244. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53, wherein the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence
set forth in SEQ ID NOS:52, 56 and 58, respectively, and the
V.sub.L region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising
the sequence set forth in SEQ ID NOS:60, 62 and 64,
respectively.
[1081] 245. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53, wherein the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence
set forth in SEQ ID NOS:52, 97 and 99, respectively, and the
V.sub.L region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising
the sequence set forth in SEQ ID NOS:60, 62 and 64,
respectively.
[1082] 246. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53, wherein the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within SEQ ID
NO:103, and the V.sub.L region comprises a CDR-L1, a CDR-L2 and a
CDR-L3 contained within SEQ ID: NO 106.
[1083] 247. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53, wherein the V.sub.H region
comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within SEQ ID
NO:130, and the V.sub.L region comprises a CDR-L1, a CDR-L2 and a
CDR-L3 contained within SEQ ID: NO 106.
[1084] 248. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53, wherein the V.sub.H region is
or comprises an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:103, and the V.sub.L region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:106.
[1085] 249. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53, wherein the V.sub.H region is
or comprises an amino acid sequence having at least at or about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO:130, and the V.sub.L region is or
comprises an amino acid sequence having at least at or about 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to SEQ ID NO:106.
[1086] 250. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53, wherein the V.sub.H region and
the V.sub.L region are or comprise the sequence set forth in SEQ ID
NOS: 103 and 106, respectively.
[1087] 251. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53, wherein the V.sub.H region and
the V.sub.L region are or comprise the sequence set forth in SEQ ID
NOS:130 and 106, respectively.
[1088] 252. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53 and 244-251, wherein the
antibody is a full length antibody.
[1089] 253. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53 and 244-251, wherein the
antibody is an antigen-binding fragment.
[1090] 254. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53 and 244-253, wherein said
anti-ROR1 antibody or antigen-binding fragment thereof is
recombinant.
[1091] 255. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53 and 244-254, wherein the V.sub.H
region and the V.sub.L region is human or is from a human
protein.
[1092] 256. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53 and 244-255, wherein the scFv is
or comprises the sequence set forth in SEQ ID NO: 109.
[1093] 257. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53 and 244-255, wherein the scFv is
or comprises the sequence set forth in SEQ ID NO: 134.
[1094] 258. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53 and 244-257, wherein said
anti-ROR1 antibody or antigen-binding fragment thereof specifically
binds to an epitope consisting of the sequence set forth in SEQ ID
NO:199 or an epitope present within the sequence set forth in SEQ
ID NO:199.
[1095] 259. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53 and 244-257, wherein the
antibody or antigen-binding fragment thereof further binds to one
or more epitopes consisting of a sequence selected from among any
one of SEQ ID NOS: 200-214 or an epitope present within a sequence
selected from among any one of SEQ ID NOS: 200-214.
[1096] 260. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53 and 244-259, wherein the
antibody or antigen-binding fragment thereof binds to human ROR1
protein with an equilibrium dissociation constant (K.sub.D) of from
about 1.times.10.sup.-11 M to about 1.times.10.sup.-7 M.
[1097] 261. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53 and 244-260, wherein the
antibody or antigen-binding fragment thereof binds to human ROR1
protein with an equilibrium dissociation constant (K.sub.D) of from
about 1.times.10.sup.-8 M to about 1.times.10.sup.-7 M.
[1098] 262. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53 and 244-260, wherein the
antibody or antigen-binding fragment thereof binds to human ROR1
protein with an equilibrium dissociation constant (K.sub.D) of from
about 5.times.10.sup.-11 M to about 1.times.10.sup.10 M.
[1099] 263. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53 and 244-262, wherein the
antibody or antigen-binding fragment thereof binds to human ROR1
protein with a dissociation rate constant (k.sub.d or k.sub.off) of
from about 1.times.10.sup.-5 1/s to about 1.times.10.sup.-2
1/s.
[1100] 264. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53 and 244-263, wherein the
antibody or antigen-binding fragment thereof binds to human ROR1
protein with a dissociation rate constant (k.sub.d or k.sub.off) of
from about 1.times.10.sup.-3 1/s to about 1.times.10.sup.-2
1/s.
[1101] 265. The anti-ROR1 antibody or antigen-binding fragment
thereof of any of embodiments 1-53 and 244-263, wherein the
antibody or antigen-binding fragment thereof binds to human ROR1
protein with a dissociation rate constant (k.sub.d or k.sub.off) of
from about 1.times.10.sup.-5 1/s to about 1.times.10.sup.-4
1/s.
[1102] 266. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177, wherein the at least a portion of a hinge
region comprises all or a portion of an IgG4 hinge region,
optionally a human IgG4 hinge region, or a variant thereof.
[1103] 267. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177, wherein the at least a portion of a hinge
region comprises all or a portion of an IgG2 hinge region,
optionally a human IgG2 hinge region, or a variant thereof.
[1104] 268. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177, wherein the spacer is or comprises the sequence
set forth in SEQ ID NO: 1.
[1105] 269. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177, wherein the spacer is or comprises the sequence
set forth in SEQ ID NO: 135.
[1106] 270. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177, wherein the spacer comprises at least a portion
of a hinge region and at least a portion of a CH3 region of an
immunoglobulin or a variant thereof.
[1107] 271. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 270, wherein the at least a portion of a CH3
region comprises all or a portion of an IgG4 CH3 and/or an IgG2
CH3, wherein the IgG4 CH3 is optionally a human IgG4 CH3 and the
IgG2 CH3 is optionally a human IgG2 CH3.
[1108] 272. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177, 270 and 271, wherein the spacer is at or about
111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,
124 or 125 amino acids in length, or has a length between any of
the foregoing.
[1109] 273. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 270-272, wherein the spacer is at or about
120 amino acids in length.
[1110] 274. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 270-273, wherein the spacer is or comprises
an amino acid sequence having at least at or about 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 3 or 138.
[1111] 275. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 270-274, wherein the spacer is or comprises
the sequence set forth in SEQ ID NO: 3 or 138.
[1112] 276. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177, wherein the spacer comprises at least a portion
of a hinge region, at least a portion of a CH2 and at least a
portion of a CH3 region of an immunoglobulin or a variant
thereof.
[1113] 277. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 276, wherein the at least a portion of a CH2
region comprises all or a portion of an IgG4 CH2 and/or an IgG2
CH2, wherein the IgG4 CH2 is optionally a human IgG4 CH2 and the
IgG2 CH2 is optionally a human IgG2 CH2.
[1114] 278. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177, 276 and 277, wherein the spacer is at or about
130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223,
224, 225, 226, 227, 228, 229 or 230 amino acids in length, or has a
length between any of the foregoing.
[1115] 279. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 276-278, wherein:
[1116] one or more of the hinge region, the CH2 region and the CH3
region comprises all or a portion of a CH2 region and all or a
portion of a CH3 region from human IgG4; or
[1117] one or more of the hinge region, the CH2 region and the CH3
region is chimeric and comprises a hinge, a CH2 region and a CH3
region from human IgG4 and human IgG2; or the spacer comprises a
IgG4/2 chimeric hinge region or a modified IgG4 hinge region
comprising at least one amino acid replacement compared to a human
IgG4 hinge; an IgG2/4 chimeric CH2 region; and an IgG4 CH3
region.
[1118] 280. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 276-279, wherein the spacer is or comprises
an amino acid sequence having at least at or about 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO:37 or 194.
[1119] 281. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 276-280, wherein the spacer is or comprises
the sequence set forth in SEQ ID NO: 37 or 194.
[1120] 282. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-281, wherein the transmembrane region is
or comprises a transmembrane domain from CD4, CD28, or CD8.
[1121] 283. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-282, wherein the transmembrane region is
or comprises a transmembrane domain from CD28, optionally a human
CD28.
[1122] 284. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-283, wherein the transmembrane domain is
or comprises SEQ ID NO: 8 or 149 or an amino acid sequence having
at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% sequence identity to SEQ ID NO: 8 or 149.
[1123] 285. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-284, wherein the intracellular signaling
region further comprises a costimulatory signaling region.
[1124] 286. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-285, wherein the anti-ROR1 chimeric
antigen receptor is or comprises the sequence set forth in SEQ ID
NO: 184, 185, 186, 187, 188 or 189 or a sequence that exhibits at
least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% sequence identity to the sequence set forth in SEQ
ID NO: 184, 185, 186, 187, 188 or 189.
[1125] 287. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-286, wherein the anti-ROR1 chimeric
antigen receptor is or comprises the sequence set forth in SEQ ID
NO: 184 or a sequence that exhibits at least 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the sequence set forth in SEQ ID NO: 184.
[1126] 288. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-286, wherein the anti-ROR1 chimeric
antigen receptor is or comprises the sequence set forth in SEQ ID
NO: 184.
[1127] 289. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-286, wherein the anti-ROR1 chimeric
antigen receptor is or comprises the sequence set forth in SEQ ID
NO: 185 or a sequence that exhibits at least 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the sequence set forth in SEQ ID NO:185.
[1128] 290. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-286, wherein the anti-ROR1 chimeric
antigen receptor is or comprises the sequence set forth in SEQ ID
NO: 185.
[1129] 291. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-286, wherein the anti-ROR1 chimeric
antigen receptor is or comprises the sequence set forth in SEQ ID
NO: 186 or a sequence that exhibits at least 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the sequence set forth in SEQ ID NO: 186.
[1130] 292. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-286, wherein the anti-ROR1 chimeric
antigen receptor is or comprises the sequence set forth in SEQ ID
NO: 186.
[1131] 293. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-286, wherein the anti-ROR1 chimeric
antigen receptor is or comprises the sequence set forth in SEQ ID
NO: 187 or a sequence that exhibits at least 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the sequence set forth in SEQ ID NO:187.
[1132] 294. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-286, wherein the anti-ROR1 chimeric
antigen receptor is or comprises the sequence set forth in SEQ ID
NO: 187.
[1133] 295. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-286, wherein the anti-ROR1 chimeric
antigen receptor is or comprises the sequence set forth in SEQ ID
NO: 188 or a sequence that exhibits at least 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the sequence set forth in SEQ ID NO: 188.
[1134] 296. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-286, wherein the anti-ROR1 chimeric
antigen receptor is or comprises the sequence set forth in SEQ ID
NO: 188.
[1135] 297. The anti-ROR1 chimeric antigen receptor of any of
embodiments 57-177 and 266-286, wherein the anti-ROR1 chimeric
antigen receptor is or comprises the sequence set forth in SEQ ID
NO: 189 or a sequence that exhibits at least 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the sequence set forth in SEQ ID NO:189.
[1136] 298. A method of treatment, comprising administering the
cell of any of embodiments 221-226 or the composition of any of
embodiments 227-231 to a subject having a disease or disorder
associated with ROR1.
[1137] 299. The cell of any of embodiments 221-226 or the
composition of any of embodiments 227-231 for use in treating a
disease or disorder associated with ROR1.
[1138] 300. Use of the cell of any of embodiments 221-226 or the
composition of any of embodiments 227-231 for the manufacture of a
medicament for treating a disease or disorder associated with
ROR1.
[1139] 301. Use of the cell of any of embodiments 221-226 or the
composition of any of embodiments 227-231 for the treatment of a
disease or disorder associated with ROR1.
[1140] 302. The method, the cell, composition, antibody or
antigen-binding fragment thereof, single chain cell surface
protein, conjugate, chimeric antigen receptor, polynucleotide or
vector for use or the use of any of embodiments 232-241 and
298-302, wherein the disease or disorder associated with ROR1 is a
cancer.
[1141] 303. The method, the cell, composition, antibody or
antigen-binding fragment thereof, single chain cell surface
protein, conjugate, chimeric antigen receptor, polynucleotide or
vector for use or the use of embodiment 302, wherein the cancer is
a ROR1-expressing cancer.
[1142] 304. The method, the cell, composition, antibody or
antigen-binding fragment thereof, single chain cell surface
protein, conjugate, chimeric antigen receptor, polynucleotide or
vector for use or the use of any of embodiments 232-241 and
298-303, wherein the cancer is associated with a ROR1-expressing
solid tumor or a ROR1-expressing hematologic malignancy.
[1143] 305. The method, the cell, composition, antibody or
antigen-binding fragment thereof, single chain cell surface
protein, conjugate, chimeric antigen receptor, polynucleotide or
vector for use or the use of any of embodiments 232-241 and
298-304, wherein the cancer is associated with a ROR1-expressing
solid tumor.
[1144] 306. The method, the cell, composition, antibody or
antigen-binding fragment thereof, single chain cell surface
protein, conjugate, chimeric antigen receptor, polynucleotide or
vector for use or the use of embodiment 304 or embodiment 305,
wherein the cancer associated with a solid tumor is selected from
the group consisting of neuroblastoma, renal cell carcinoma, colon
cancer, colorectal cancer, breast cancer, epithelial squamous cell
cancer, melanoma, myeloma, stomach cancer, brain cancer, lung
cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver
cancer, bladder cancer, prostate cancer, testicular cancer, thyroid
cancer, uterine cancer, adrenal cancer and head and neck
cancer.
[1145] 307. The method, the cell, composition, antibody or
antigen-binding fragment thereof, single chain cell surface
protein, conjugate, chimeric antigen receptor, polynucleotide or
vector for use or the use of embodiment 306, wherein the lung
cancer is a non-small cell lung cancer (NSCLC), lung
adenocarcinoma, adenocarcinoma, squamous cell carcinoma, small cell
carcinoma, and atypical carcinoid.
[1146] 308. The method, the cell, composition, antibody or
antigen-binding fragment thereof, single chain cell surface
protein, conjugate, chimeric antigen receptor, polynucleotide or
vector for use or the use of embodiment 307, wherein the lung
cancer is a NSCLC.
[1147] 309. The method, the cell, composition, antibody or
antigen-binding fragment thereof, single chain cell surface
protein, conjugate, chimeric antigen receptor, polynucleotide or
vector for use or the use of embodiment 306, wherein the breast
cancer is a triple negative breast cancer (TNBC).
[1148] 310. The method, the cell, composition, antibody or
antigen-binding fragment thereof, single chain cell surface
protein, conjugate, chimeric antigen receptor, polynucleotide or
vector for use or the use of any of embodiments 232-241 and
298-304, wherein the cancer is associated with a ROR1-expressing
hematologic malignancy.
[1149] 311. The method, the cell, composition, antibody or
antigen-binding fragment thereof, single chain cell surface
protein, conjugate, chimeric antigen receptor, polynucleotide or
vector for use or the use of embodiment 310, wherein the
hematologic malignancy is selected from the group consisting of B
cell leukemia, lymphoma, B cell chronic lymphocytic leukemia (CLL),
acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL),
Burkitt's Lymphoma or mantle cell lymphoma (MCL).
VIII. EXAMPLES
[1150] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
invention.
Example 1: Generation and Assessment of Anti-ROR1 Antibodies
[1151] Exemplary anti-receptor tyrosine kinase-like orphan receptor
1 (ROR1) antibodies that specifically bind to ROR1-expressing cells
were generated by immunization of genetically engineered mice and
assessed.
[1152] A. Antibody Generation
[1153] Mice that were genetically modified to produce antibodies
containing fully human antibody variable regions (Trianni, Inc.,
San Francisco, Calif.) were immunized with recombinant
extracellular domain (ECD) of human ROR1 fused to a histidine tag.
The spleen, lymph nodes and bone marrow were harvested, and ROR1
specific memory B cells and plasma cells were enriched by
immunoaffinity-based selection.
[1154] Cell populations from the enriched fractions were subject to
high-throughput single-cell paired sequencing of antibody variable
heavy (V.sub.H) chain and variable light (V.sub.L) chain. High
throughput single cell antibody sequencing was performed as
generally described in WO2012/048340, WO2012/048341, WO2016/044227
and WO2016/176322. The sequencing methods employed single-cell
droplets, with droplet and molecular barcodes, to identify
individual pairs of antibody V.sub.H and V.sub.L sequences at a
single-cell level, for each of a large number of single cells
present in a population of cells. Antibody V.sub.H/V.sub.L pair
sequences were selected for synthesis and polynucleotides encoding
each of the paired candidates were synthesized as a single chain Fv
fragment (scFv), in either a V.sub.H and V.sub.L chain orientation
(V.sub.H-V.sub.L) or a V.sub.L-V.sub.H orientation.
[1155] B. Anti-ROR1 Chimeric Antigen Receptor (CAR) Generation
[1156] The synthesized scFv-encoding sequences were cloned into an
exemplary polynucleotide construct to generate candidate
polynucleotides encoding chimeric antigen receptors (CARs)
containing the candidate scFvs as the antigen-binding domains.
Specifically, the polynucleotide CAR constructs contained nucleic
acid sequences encoding a signal peptide; a candidate anti-ROR1
scFv; a spacer from among three exemplary spacers: a spacer
containing a modified IgG4/IgG2-hinge C.sub.H2-CH3 (SEQ ID NO:194),
a spacer containing a modified IgG4 hinge-C.sub.H3 (SEQ ID NO:138),
or a spacer containing a modified IgG4 hinge region (SEQ ID
NO:135); a human CD28 transmembrane domain; a human 4-1BB
intracellular signaling region; and a human CD3-zeta intracellular
signaling region. The constructs also contained a downstream T2A
ribosomal skip elements (SEQ ID NO: 6) between the CAR-encoding
sequences and sequences encoding a green fluorescent protein for
use as a transduction marker. Polynucleotides encoding the CARs
were cloned into a lentiviral expression vector for transduction of
T cells.
[1157] C. Assessment of Candidate Anti-ROR1 CARs
[1158] Approximately 400 candidate anti-ROR1 CAR-encoding
constructs were generated as described above and viral preparations
containing constructs encoding the various candidate anti-ROR1 CARs
were individually introduced into a Jurkat T cell line containing a
Nur77 knock-in reporter (see e.g. WO 2019/089982). The Nur77
knock-in cell line contained nucleic acid sequences encoding a
reporter molecule (e.g., a red fluorescent protein) knocked-in at
the endogenous Nur77 locus, which is an immediate-early response
gene induced by stimulation of signal from the T cell receptor
and/or via molecules containing immunoreceptor tyrosine-based
activation motif (ITAM). The Jurkat reporter cells were assessed
for cell surface expression of the CAR, antigen-dependent and
antigen-independent signaling and cross-reactivity to a related but
distinct antigen ROR2.
[1159] After transduction, the reporter cells were evaluated for
the expression of red fluorescent protein after co-culture with
target cells, including K562 human myelogenous leukemia cells (ROR1
negative), K562 cells transduced to express a high level of ROR1,
K562 cells transduced to express a low level of ROR1, and K562
cells expressing the non-specific antigen ROR2. To further assess
antigen-independent activity, transduced reporter cells were
cultured alone in the absence of antigen-expressing target cells.
The transduced reporter cells were also assessed for GFP expression
(transduction marker), and for binding of soluble recombinant
ROR1-Fc (soluble human ROR1 fused to an Fc region of an IgG).
[1160] Jurkat reporter cells transduced with candidate anti-ROR1
CARs exhibited varying levels of antigen-specific activity, as
indicated by reporter expression after co-culture with
ROR1-expressing target cells expressing high or low levels of ROR1;
varying levels of antigen-independent (tonic) signaling, as
indicated by reporter expression after co-culture with K562 cells
not expressing ROR1 or after culture without antigen-expressing
target cells; varying levels of non-specific activity, as indicated
by reporter expression after co-culture with cells expressing the
non-specific antigen ROR2; and varying levels of binding by soluble
ROR1-Fc. Candidate CARs that exhibited high ROR1 antigen-dependent
activity, low tonic signaling, high ROR1-Fc binding and low ROR2
cross-reactivity were selected for assessment in primary human T
cells.
Example 2: Assessment of CAR Expression and Function in Primary T
Cells
[1161] The expression and function of selected anti-ROR1 CARs,
described in Example 1, were assessed in primary human T cells.
[1162] CD4+ and CD8+ T cells were isolated by immunoaffinity-based
enrichment from leukapheresis samples of one of three human donors.
Isolated CD4+ and CD8+ T cells were mixed at approximately 1:1
ratio, stimulated and transduced with lentiviral preparations
encoding one of the anti-ROR1 CARs selected as described in Example
1 (designated individually one of A-Z and A1-M1) and cultivated
under conditions for expansion. The three human donors varied in
the percentage of T cell subsets present in the leukapheresis
sample (e.g. less than 15% naive T cells, about 40-60% naive T
cells or about 20% effector memory RA T cells (T.sub.EMRA)),
representing variability in T cell subsets among disease subjects.
As controls, primary T cells also were similarly stimulated and
transduced with lentiviral preparations encoding an anti-CD19 CAR,
a reference anti-ROR1 CAR (a rabbit-derived reference antibody R12,
scFv set forth in SEQ ID NO:143) or one of two anti-ROR1 CARs
obtained from a different source (designated N1, 01).
[1163] The CAR-expressing primary T cells were stimulated with
plate-bound ROR1-Fc, and after 24 hours were assessed by flow
cytometry for intracellular levels of IFN-.gamma., TNF-.alpha. and
IL-2 by intracellular cytokine staining (ICS). The percentage of
cells producing IFN-.gamma., TNF-.alpha. and IL-2, and the level of
production per cell (as determined by the mean fluorescence
intensity (MFI)) were determined. After stimulation, the
CAR-expressing primary T cells were also stained for cell surface
expression of T cell activation markers (e.g., CD25), costimulatory
markers, markers indicative of certain T cell subtypes, such as
memory cell subtypes, other T cell markers, e.g., CD4 and CD8,
ROR1-Fc binding, and assessed for expression of the GFP
transduction marker.
[1164] CAR-expressing primary T cells also were stimulated by
co-culture with ROR1-expressing triple-negative human breast cancer
cell line (MDA-MB-231 (ATCC.RTM. HTB-26.TM.), at an effector:target
(E:T) ratio of 1:4, and assessed for cytotoxicity and cytokine
production. The MDA-MB-231 cell line was labeled with NucLight Red
(NLR) to permit tracking of target cells by microscopy. Cytolytic
activity was assessed by measuring the loss of viable target cells
over a period of between 0 and 72 hours, as determined by changes
in red fluorescent signal. The production of cytokines IFN-.gamma.,
TNF-.alpha. and IL-2 in the supernatant was also assessed.
[1165] As shown in FIG. 1, certain candidate anti-ROR1
CAR-expressing T cells exhibited comparable or greater relative
intracellular TFN-.gamma., TNF-.alpha. or IL-2 expression levels by
ICS in GFP+ CD4+ T cells compared to the levels of each cytokine in
cells expressing the reference anti-ROR1 (R12) CAR, after 24 hours
stimulation with plate-bound recombinant ROR1-Fc. In the co-culture
assay with MDA-MB-231 target cells expressing ROR1, certain
selected candidates also exhibited comparable or greater
IFN-.gamma., TNF-.alpha. or IL-2 secretion compared to cytokine
secretion in cells expressing the reference anti-ROR1 (R12) CAR, as
shown in FIG. 2. Fewer anti-ROR1 candidates exhibited comparable or
greater cytokine secretion compared to reference anti-ROR1 (R12)
CAR in the co-culture assay, compared to the number of anti-ROR1
candidates that showed comparable or greater cytokine expression
levels by ICS.
[1166] These results showed varied responses by primary human T
cells engineered to express the candidate anti-ROR1 CARs after
stimulation by recombinant antigen or antigen-expressing target
cells.
Example 3: Polynucleotide Optimization of Selected Anti-ROR1
Chimeric Antigen Receptors (CARs)
[1167] Among the candidate anti-ROR1 CARs, six (6) candidate CARs
were further selected based on results of assessment of functional
activity in engineered primary human T cells from the 3 different
donors as described in Example 2. The constructs encoding the
selected candidate CARs were subject to codon-optimization (CO) and
assessed for potential splice sites and modified in a conservative
manner, such as by not altering the encoded amino acid sequence,
including elimination of potential predicted splice sites (SSE) in
the polynucleotide construct encoding the CAR. The constructs were
further modified to replace the GFP transduction marker with a
truncated receptor as a surrogate transduction marker for CAR
expression.
[1168] Table E1 lists sequence identifiers (SEQ ID NO:)
corresponding to amino acid (aa) sequences of the of the
antigen-binding domains of the selected candidate CARs, including
amino acid sequences of the corresponding variable heavy (V.sub.H)
chain and variable light (V.sub.L) chain, and complementarity
determining regions (CDRs, by Kabat numbering) of each chain. The
sequence identifier (SEQ ID NO:) of the V.sub.H and V.sub.L chain
of the reference R12 CAR also is set forth in Table E1. The
exemplary CARs contained an scFv having the V.sub.H-V.sub.L
orientation.
TABLE-US-00004 TABLE E1 Sequence identifier (SEQ ID NO:) for
Exemplary Candidate CARs Nucleic Acid (codon optimized and splice
site eliminated) Amino Acid (CDR Kabat numbering) CAR scFv #
V.sub.H V.sub.L scFv spacer V.sub.H CDR-H1 CDR-H2 CDR-H3 VL CDR-L1
CDR-L2 CDR-L3 scFv spacer A ROR1-2 120 123 126 136 121 82 86 88 124
90 92 94 127 135 F ROR1-1 111 114 117 136 112 67 71 73 115 75 77 79
118 135 G ROR1-1 111 114 117 139 112 67 71 73 115 75 77 79 118 138
I ROR1-3 102 105 108 139 103 52 56 58 106 60 62 64 109 138 R ROR1-2
120 123 126 139 121 82 86 88 124 90 92 94 127 138 B1 ROR1-4 129 131
133 139 130 52 97 99 106 60 62 64 134 138 R12 142
[1169] To assess activity of CARs encoded by the polynucleotides,
CD4+ and CD8+ T cells isolated by immunoaffinity-based enrichment
from leukapheresis samples from two (2) human donor subjects were
stimulated and transduced with lentiviral preparations encoding one
of the 6 candidate CARs or the reference anti-ROR1 (R12) CAR as a
control.
[1170] The cells expressing the candidate CARs were co-cultured
with a ROR1-expressing human non-small cell lung cancer cell line
H1975 (ATCC.RTM. CRL-5908.TM.) labeled with NucLight Red (NLR), and
cytotoxicity and cytokine production was assessed. Cytolytic
activity was assessed by measuring the loss of viable target cells
over a period of between 0 and 70 hours, as determined by changes
in red fluorescent signal. The production of cytokines TNF-.alpha.,
TNF-.alpha. and IL-2 in the supernatant was also assessed after 70
hours of co-culture. As controls, cultures of target cells only and
co-cultures of mock cells (not expressing a CAR) with the target
cells were assessed.
[1171] As shown in FIGS. 3A-3B, all of the candidate anti-ROR1 CARs
tested showed similar in vitro cytotoxic activity against the H1975
target cells. As shown in FIGS. 4A-4B, all of the candidate
anti-ROR1 CARs tested produced IFN-.gamma., TNF-.alpha. and IL-2 at
various levels after in vitro co-culture with H1975 target cells.
The results showed that after codon optimization and splice site
elimination, all test constructs demonstrated cytotoxicity in
co-culture with the H1975 (NSCLC) cell line. Among the tested CARs
in this assay, cells expressing anti-ROR1 CAR-A, anti-ROR1 CAR-F
and anti-ROR1 CAR-G generally exhibited among the highest cytokine
production, which, for the various cytokines assessed, was similar
to or greater than cytokine production of the cytokines from cells
expressing the reference anti-ROR1 (R12) CAR. In particular, cells
expressing anti-ROR1 CAR-F exhibited the highest IL-2 production
compared to other candidate anti-ROR1 CARs and the reference
anti-ROR1 (R12) CAR. The results were consistent with robust in
vitro functional activity of the candidate CARs in co-culture with
H1975 target cells.
[1172] The production of cytokines IL-2 in the supernatant was also
assessed after co-culture with other ROR1 expressing target cell
lines, including MDA-MB-231 human triple negative breast cancer
cells, A549 human epithelial lung carcinoma cells and BT-549 human
epithelial breast ductal carcinoma cells, at an E:T ratio of 1:4.
As shown in FIG. 4C, all of the candidate anti-ROR1 CARs tested
produced IL-2 at various levels after in vitro co-culture with
MDA-MB-231, A549 and BT-549 target cells. The results showed that
cells expressing anti-ROR1 CAR-F exhibited the highest IL-2
production compared to other candidate anti-ROR1 CARs and the
reference anti-ROR1 (R12) CAR, when co-cultured with all of the
various target cells.
Example 4: Anti-Tumor Effect of Anti-ROR1 CAR-Expressing T Cells
after Adoptive Transfer In Vivo in an Animal Model
[1173] The anti-tumor effects of exemplary engineered anti-ROR1
CAR-expressing primary human T cells were assessed by monitoring
tumors following adoptive transfer of cells in tumor-bearing animal
models, including a H1975 human non-small cell lung cancer
xenograft model and a MDA-MB-231 human triple negative breast
cancer xenograft model. The mice were administered a preparation of
engineered primary human T cells generated from one of the human
donors, expressing one of anti-ROR1 CARs-A, -F, -G, -I, -R and -B1
as listed in Table E1 or a reference anti-ROR1 (R12) CAR, and that
had been assessed in the in vitro assays described in Example
3.
[1174] A. H1975 Non-Small Cell Lung Cancer Model
[1175] 1. Study 1
[1176] Eighty (80) NOD.Cg.Prkdc.sup.scidIL2rg.sup.tm/WjlSzJ (NSG)
mice were each injected subcutaneously with approximately
5.times.10.sup.6 H1975 Non-Small Cell Lung Cancer cells. On day 16
following tumor engraftment, five (5) mice in each group received a
single intravenous (i.v.) injection of engineered primary human T
cells expressing one of anti-ROR1 CAR-A, -F, -G, -I, -R and -B1 or
the reference anti-ROR1 (R12) CAR, at a dose of 1.times.10.sup.6
cells (low dose) or 3.times.10.sup.6 cells (high dose). As a
control, mice were administered 3.times.10.sup.6 cells not
expressing a CAR (mock) or were untreated. Survival, tumor volume
and number of circulating CAR.sup.+ cells in the blood were
assessed over approximately 65 days.
[1177] Anti-tumor activity of the adoptively transferred anti-ROR1
CAR+ T cells was monitored by determining the tumor volume every 3
to 6 days post administration. FIG. 5A (low dose) and FIG. 5B (high
dose) depict mean tumor volume of all treated mice; in this
depiction, tumor curves were terminated after the first mouse of a
group succumbed to disease. Results from all individual mice are
shown in FIG. 5C (low dose) or FIG. 5D (high dose). As shown in
FIGS. 5A-5D, primary human T cells expressing the candidate
anti-ROR1 CARs showed varying levels of anti-tumor activity in the
H1975 mouse model. Primary human T cells expressing anti-ROR1 CAR-F
exhibited the highest anti-tumor activity after a high dose
administration, as shown by reduction in tumor volume, of tested
anti-ROR1 CARs, including the anti-ROR (R12) reference CAR.
Specifically, administration of anti-ROR1 CAR-F expressing cells
resulted in mean tumor regression (below baseline) up to 52 days,
compared to 6 days for the reference R12 CAR-expressing cells. 80%
of H1975 tumor-bearing mice (4 of 5) treated with the high dose of
anti-ROR1 CAR-F expressing cells exhibited complete and durable
tumor clearance, whereas no animals treated with the high dose of
the reference R12 CAR-expressing cells exhibited complete tumor
clearance. At the low dose, 3 of 5 animals administered anti-ROR1
CAR-F expressing cells exhibited tumor regression below baseline up
to 17 days, whereas only 1 of 5 animals administered R12
CAR-expressing cells exhibited tumor regression below baseline.
[1178] Survival of mice treated as described above was assessed.
Kaplan-Meier survival curves of each of the groups are shown in
FIGS. 6A-6B. As shown, administration of primary human T cells
expressing the candidate CARs showed varying effects on survival of
the treated mice. In this model, mice administered primary human T
cells expressing anti-ROR1 CAR-F or anti-ROR1 CAR-A exhibited
survival that was similar to or longer than survival of the
reference anti-ROR1 (R12) CAR at one or both of the tested doses.
Mice administered cells expressing anti-ROR1 CAR-F exhibited the
longest survival with over 100% of mice surviving at 65 days
following the initiation of administration of CAR-expressing cells
at the highest dose in this model.
[1179] The average number of CD4+ and CD8+ CAR-expressing cells in
the blood of each mouse was determined at day 10 and day 24 after
administration of CAR-expressing T cells, at the low dose (FIGS. 7A
and 7B) or high dose (FIGS. 7C and 7D). The average number of CD3+
CAR-expressing cells (CD45+ CD3+ CAR+) in the blood of each mouse
also was determined at day 10 and day 24 after administration of
CAR-expressing T cells, at the low dose (FIG. 7E) or high dose
(FIG. 7F). As shown in FIGS. 7A-7F, a higher average CAR+ T cell
number was observed in mice administered a higher dose of CAR+ T
cells. T cells expressing anti-ROR1 CAR-F exhibited robust
expansion following administration at both the low and high doses
in this model. At 10 days post-infusion, the mean count of CAR-F
expressing cells was 226.5 and 13.1 cells/.mu.l of blood for the
high and low doses, respectively, compared to the mean count of R12
expressing cells of 7.6 and 2.5 cells/.mu.l of blood for the high
and low doses, respectively. At 24 days post-infusion, the mean
count of CAR-F expressing cells was 471.8 and 44.0 cells/0 of blood
at the high and low doses, respectively, compared to the count of
R12 expressing cells of 27.6 and 4.2 cells/0 of blood for the high
and low doses, respectively.
[1180] 2. Study 2
[1181] In a different study using the same H1975 mouse model,
sixty-four (64) mice with engrafted H1975 tumors were staged based
on measurement of tumor volume and administered a single
intravenous (i.v.) injection of engineered primary human T cells
expressing anti-ROR1 CAR-F, anti-ROR1 CAR-A, or reference anti-ROR1
(R12) CAR, at a dose of 1.times.10.sup.6 cells (low dose) or
3.times.10.sup.6 cells (high dose), eight (8) mice in each group.
As a control, mice were administered 3.times.10.sup.6 cells not
expressing a CAR (mock) or were untreated. Tumor volume was
measured up to approximately 70 days, and circulating CAR+ T cell
number and survival were measured up to approximately 40 days.
[1182] The changes in the mean tumor volume in mice that were
administered anti-ROR1 CAR-F, CAR-A or the R12 reference CAR at the
low or the high dose are shown in FIGS. 8A and 8B, respectively.
The changes in the mean tumor volume in individual mice are shown
in FIG. 8C. Administration of anti-ROR1 CAR-F expressing cells
resulted in mean tumor regression (below baseline) at 15 days,
compared to administration of reference R12 CAR-expressing cells,
which did not exhibit mean tumor regression below baseline (FIG.
8B). 25% of H1975 tumor-bearing mice (2 of 8) treated with the high
dose of anti-ROR1 CAR-F expressing cells exhibited complete and
durable tumor clearance, whereas 12.5% of mice (1 of 8) treated
with 3 the high dose of the reference R12 CAR-expressing cells
exhibited complete tumor clearance.
[1183] Mice were monitored for tumor growth and body condition at
least twice weekly for 69 days post CAR T cell transfer and were
euthanized when tumor volume exceeded 1500 mm.sup.3, when
experiencing tumor ulceration, when displaying signs of severe
xenogeneic graft versus host disease (xGvHD), or when otherwise
moribund. The survival of mice in each group are shown in FIGS.
9A-9B. Median survival in untreated H1975 xenograft mice were left
or administered mock treated cells was 20 days. Median survival of
animals administered the high or low dose of R12 CAR T cells, was
33 and 29 days, respectively. Median survival in mice administered
the anti-ROR1 CAR-F survived to 37.5 days for the low dose, and
median survival was undetermined at the high dose, with 62.5% (5 of
8) animals remaining alive at study end.
[1184] The average CD4+ and CD8+ CAR+ T cell count per microliter
of blood at days 7, 14 and 21 after administration are shown in
FIGS. 10A-10D. Consistent with the results in the study described
above, the mean count of CAR-F expressing cells was highest
compared to the meant count of the other CAR-expressing cells,
particularly at day 21 and/or among CD4+ T cells (FIG. 10A and FIG.
10C), or at the high dose (FIG. 10C and FIG. 10D). At 7, 14, and 21
days post-administration of the high dose, the anti-ROR1 CAR-F
expressing T cells showed mean counts of 37.6, 105.6, and 46.6
cells/.mu.l of blood, respectively, whereas the reference
R12-expressing cells showed mean counts of 28.9, 39.8, and 20.3
cells/0 of blood. At days 7, 14, and 21 post-administration of the
lower dose, the anti-ROR1 CAR-F expressing cells showed mean counts
of 2.2, 55.4, and 6.9 cells/.mu.l of blood, respectively, whereas
the reference R12-expressing cells showed mean counts of 3.1, 40.8,
and 5.5 cells/.mu.l of blood, respectively.
[1185] FIGS. 11A and 11B depict the number of CD4+ and CD8+ CAR+ T
cells present in the tumor at 14 days after administration. The
number of CAR+ cells in the tumor at day 14 were similar between
the CARs tested.
[1186] 3. Summary
[1187] As shown, mice with H1975 lung adenocarcinoma xenograft
tumors administered anti-ROR1 CAR-F-expressing cells exhibited
higher anti-tumor activity, as indicated by tumor volume reduction,
longer survival, and increased number of circulating CAR+ T cells
compared to mice administered the reference anti-ROR1 R12 CAR. The
anti-tumor activity, survival and CAR+ T cell number was similar in
mice administered anti-ROR1 CAR-A-expressing cells compared to
anti-ROR1 R12 CAR-expressing cells.
[1188] B. MDA-MB-231 Triple Negative Breast Cancer Model
[1189] Sixty-four (64) NOD.Cg.Prkdc.sup.scidIL2rg.sup.tm1lWjl/SzJ
(NSG) mice were each injected subcutaneously with 1.times.10.sup.6
MDA-MB-231 cells expressing firefly luciferase. On day 7 following
tumor engraftment, eight (8) mice in each group received a single
intravenous (i.v.) injection of engineered primary human T cells
expressing anti-ROR1 CAR-A, anti-ROR1 CAR-F, or reference anti-ROR1
(R12) CAR, at a dose of 1.times.10.sup.6 cells (low dose) or
3.times.10.sup.6 cells (high dose). As a control, mice were
administered 3.times.10.sup.6 T cells not expressing a CAR (mock)
or were untreated. Bioluminescence imaging of tumor burden and flow
cytometric analysis of the number of circulating CAR+ cells in the
blood were assessed over approximately 100 days.
[1190] Anti-tumor activity of the adoptively transferred
CAR-expressing T cells was monitored by bioluminescence imaging
every 4 to 7 days post CAR-T cell administration. For
bioluminescence imaging, mice received intraperitoneal (i.p.)
injections of luciferin substrate (PerkinElmer, Waltham, Mass.)
resuspended in PBS (approximately 150 mg/kg body weight). Mice were
anesthetized and imaged essentially as described in WO2015/095895.
The total flux (photon/s) was determined at each time point.
[1191] The results for bioluminescence images from treated mice and
the mean measured total flux (p/s) are shown in FIGS. 12B and 13A,
respectively, following administration of the low dose of
CAR-expressing cells; or in FIGS. 12C and 13B, respectively,
following administration of the high dose of CAR-expressing cells.
FIG. 12A depicts bioluminescence images of untreated mice or mice
administered mock T cells. FIG. 13C depicts the measured total flux
(p/s) of individual mice.
[1192] The changes in the mean tumor volume at the low dose or high
dose are shown in FIGS. 14A and 14B, respectively. As shown, mice
administered cells expressing anti-ROR1 CAR-F, at both the low and
high doses, were observed to generally have a lower degree of
bioluminescence signal, indicating a reduction in tumor growth over
time and/or a lower degree of tumor growth in the treated animals,
compared to mice administered cells expressing the reference
anti-ROR1 (R12) CAR. Mice administered cells expressing anti-ROR1
CAR-A, at both the low and high doses, were observed to generally
have similar bioluminescence signal compared to mice administered
cells expressing the reference anti-ROR1 (R12) CAR.
[1193] The changes in the mean tumor volume following
administration of T cells expressinganti-ROR1 CAR F and reference
CAR R12, up to a further time point in the same study are shown for
the high dose and low dose in FIGS. 14C and 14D, respectively.
Results of tumor volume for individual treated mice at the high
dose or low dose for each treated condition are shown in FIG. 14E.
Administration of anti-ROR1 CAR-F expressing cells resulted in mean
tumor regression (below baseline, as measured by BLI) for at least
35 days, when BLI measurements terminated. The reference R12 CAR
did not exhibit mean tumor regression below baseline, at either the
high dose (FIGS. 14C and 14E) or the low dose (FIGS. 14D and 14E).
By subcutaneous tumor volume, 50% of mice (4 of 8) administered
anti-ROR1 CAR-F expressing cells at the high dose exhibited
complete and durable tumor clearance, whereas only 12.5% of mice (1
of 8) treated with the high dose of R12 CAR-expressing cells
exhibited complete tumor clearance (FIGS. 14C and 14E). At the low
dose, 25% (2 of 8) of animals treated with anti-ROR1 CAR-F
expressing cells exhibited complete tumor clearance, whereas no
animals treated with reference R12 CAR-expressing cells at the low
dose resulted in complete tumor clearance (FIGS. 14D and 14E).
[1194] Mice were monitored for tumor growth and body condition at
least twice weekly for 97 days post CAR T cell transfer and were
euthanized when tumor volume exceeded 1500 mm.sup.3, when
experiencing tumor ulceration, when showing signs of severe xGvHD
(e.g., severe dermatitis and/or 20% weight loss attributed to
xGvHD), or when otherwise moribund. Survival of mice treated as
described above was assessed. Kaplan-Meier survival curves of each
of the groups are shown in FIGS. 15A-15B. Median survival for
untreated mice was 61 days. At the high dose, median survival was
undetermined for mice administered the anti-ROR1 CAR-F expressing
cells, R12 reference CAR expressing cells or mock treated cells, as
62.5% (5 of 8) of animals administered mock treated cells survived
at study end, and there were no deaths in either groups
administered CAR T cells (FIG. 15A). At the low dose, median
survival for the mice administered the R12 CAR-expressing cells at
the low dose was 96 days, with only 3 of 8 animals remaining alive
at study end, whereas 100% survival (8 of 8) was observed in mice
administered CAR-F expressing cells (FIG. 15B).
[1195] The average number of CD4+ and CD8+ CAR-expressing cells in
the blood of the animal were determined at days 7, 14, 21 and 30
after administration of CAR-expressing T cells at the low dose
(FIGS. 16A-16B) or high dose (FIGS. 16C-16D). The average number of
CD3+ CAR-expressing cells (CD45+ CD3+ CAR+) in the blood of each
mouse administered cells expressing anti-ROR1 CAR-F or the R12
reference CAR also was determined at days 7, 14, 21 and 30 are
shown in FIG. 16E. At the high dose, the mean count of CAR-F
expressing T cells was 5.0, 19.4, 50.7 and 119.2 cells/.mu.l of
blood for days 7, 14, 21, and 30, respectively, compared to the
mean count of R12 expressing T cells of 6.0, 5.4, 3.9, and 5.4
cells/.mu.l of blood. Administration of the low dose also showed a
similar expansion, with the mean count of CAR-F expressing cells of
1.4, 4.9, 20.9 and 58.5 cells/.mu.l of blood for days 7, 14, 21,
and 30, respectively, compared to the mean count of R12 expressing
cells of 1.3, 0.6, 0.9, and 0.4 cells/.mu.l of blood. As shown,
cells expressing anti-ROR1 CAR-F exhibited high expansion in the
MDA-MB-231 mouse model, when administered at both high and low
doses. In particular, anti-ROR1 CAR F-expressing cells exhibited
substantially high expansion starting at day 14 continuing to day
30 after administration.
[1196] C. Conclusion
[1197] The results supported high anti-tumor activity and T cell
expansion, and extended survival of mice administered anti-ROR1
CAR-F, using two different in vivo mouse tumor models, including
the triple negative breast cancer xenograft tumor model. By most
measurements, the anti-ROR1 CAR-F-expressing cells showed
improvements compared to the anti-ROR1 reference R12 CAR.
Example 5: Anti-ROR1 CAR-Expressing T Cells Generated from Human
Subjects with Chronic Lymphocytic Leukemia (CLL)
[1198] Engineered anti-ROR CAR-expressing primary T cells were
generated from human subjects with chronic lymphocytic leukemia
(CLL) and assessed for cytotoxicity and cytokine production in
vitro.
[1199] A. Generation of Anti-ROR1 CAR-Expressing Cells from
Subjects with CLL
[1200] Primary T cells from human subject with Chronic Lymphocytic
Leukemia (CLL) were engineered to express the exemplary anti-ROR1
CAR, anti-ROR1 CAR-F, described in Example 3 above.
[1201] CD4+ and CD8+ T cells were isolated by immunoaffinity-based
enrichment from leukapheresis samples of one of two human donors
with CLL. Isolated CD4+ and CD8+ T cells were mixed at
approximately 1:1 ratio, stimulated and transduced with lentiviral
preparations encoding anti-ROR1 CAR-F as described in Example 3, an
anti-CD19 CAR as a control or a reference anti-ROR1 CAR (R12), and
cultivated under conditions for expansion. CD4- and CD8-depleted
leukapheresis samples were assessed for CD19 and ROR1 expression
levels and used as target cell population in the assays. For one of
the subjects with CLL (CLL donor 1), ROR1 expression level was low,
nearly as low as a K562 human chronic myelogenous leukemia (CML)
cell line engineered to express ROR1 in response to the
tetracycline derivative doxycycline, without addition of
doxycycline (see Example 9).
[1202] B. Cytolytic Activity and Cytokine Production
[1203] Cytolytic activity of the primary T cells from subjects with
CLL expressing the exemplary anti-ROR1 CAR F were assessed.
[1204] In one experiment, target cells obtained from the subjects
(CD4- and CD8-depleted leukapheresis samples) were labeled with
Rapid Red cell labeling reagent to permit tracking of target cells
by microscopy, and incubated with the CAR-expressing cells at an
E:T ratio of 2.5:1 and 0.25:1, or cultured with mock cells or
cultured alone as controls. Cytolytic activity was assessed by
measuring the loss of red fluorescent signal over a period of
between 0 and 60 hours, compared to controls.
[1205] As shown in FIG. 17, for both donors and both E:T ratios,
red fluorescent signal from the labeled target cells were lost more
rapidly when co-cultured with CAR-expressing cells, compared to the
expected loss from normal cell division observed in the target
cells alone or co-culture with mock transduced cells. A greater
loss of target cells was observed following co-culture of the
target cells with anti-ROR1 CAR-F-expressing cells compared to
anti-ROR1 R12-expressing cells, and was comparable to the loss of
target cells following co-culture with anti-CD19 CAR-expressing
cells.
[1206] In a different experiment, the loss of ROR1 expressing
target cells was assessed by flow cytometry. For this, engineered
CAR-expressing cells were labeled with CellTrace.TM. Violet (CTV)
cell proliferation reagent, and co-cultured with target cells from
the subjects with CLL at an E:T ratio of 2.5:1 and 0.25:1. On day
4, supernatants were collected to evaluate cytokine production
(IFN-.gamma., TNF-.alpha. and IL-2) and the cells were collected to
assess CTV levels by flow cytometry.
[1207] As shown in FIG. 18A, the number of ROR1+ target cells
co-cultured with anti-ROR1 CAR F expressing cells generated from
subjects with CLL were reduced substantially compared to target
cells cultured alone or target cells co-cultured with mock
transduced T cells. The reduction was greater than the reduction
observed with a co-culture with R12-expressing cells, at both E:T
ratios for both donors. As shown in FIGS. 18B-18C, anti-ROR1 CAR F
expressing cells generated from subjects with CLL divided (FIG.
18B) and produced IFN-.gamma., TNF-.alpha. and IL-2 (FIG. 18C) in a
co-culture with target cells from the same subject. Proliferation
and cytokine production was similar to or higher than from
R12-expressing cells tested in the same condition.
[1208] The results were consistent with an observation that
engineered T cells expressing the exemplary anti-ROR1 CAR F were
successfully generated from primary T cells from human subjects
that has CLL. The cells were viable, exhibited cytolytic activity,
produced cytokines and proliferated when incubated with
ROR1-expressing target cells from the same subject, in some cases
even when the target cells expressed low levels of ROR1. The
results supported that cells expressing the exemplary anti-ROR1 CAR
F can be generated with cells from subjects with CLL, previously
reported as having difficulties in generating engineered
CAR-expressing cells (see, e.g., Gorgun et al., J Clin Invest. 2005
July; 115(7):1797-805; Ramsay et al., J Clin Invest. 2008 July;
118(7):2427-37; Riches et al., Discov Med. 2013 December;
16(90):295-302). The results also showed that the exemplary
anti-ROR1 CAR expressing cells are effective in killing target
cells that express even low levels of ROR1, demonstrating a high
sensitivity. By most measurements, the anti-ROR1 CAR-F-expressing
cells showed improvements compared to cells expressing the
anti-ROR1 reference R12 CAR.
Example 6: Anti-Tumor Effect of Anti-ROR1 CAR-Expressing T Cells
after Adoptive Transfer In Vivo in an Animal Model of a B Cell
Malignancy
[1209] The anti-tumor effects of exemplary engineered anti-ROR1
CAR-expressing (CAR.sup.+) primary human T cells were assessed by
monitoring tumors following adoptive transfer of cells in an animal
model of a disseminated human B cell malignancy. The mice were
administered a preparation of engineered primary human T cells
generated from human donors, expressing an exemplary anti-ROR1
CAR-F, compared to a reference anti-ROR1 (R12) CAR or an anti-CD19
CAR, as described in Example 3 above.
[1210] NOD.Cg.Prkdc.sup.scidIL2rg.sup.tm1Wjl/SzJ (NSG) mice were
injected intravenously with 1.0.times.10.sup.6 firefly luciferase
and green fluorescent protein (FfLuc-GFP)-expressing human mantel
cell lymphoma (MCL) JeKo-1 cells on day 0. Tumor burden was
assessed by bioluminescent imaging (BLI), by detecting
bioluminescence minutes after mice were injected intraperitoneally
with 3 mg of D-Luciferin. On day 6, mice were randomized into
groups to balance tumor burden. On day 7, mice were administered
engineered primary human T cells generated from a human donor
expressing the exemplary anti-ROR1 CAR-F or the reference anti-ROR1
(R12) CAR, at a dose of 1.times.10.sup.6 cells (low dose) or
3.times.10.sup.6 cells (high dose) per mouse. As controls, mice
either did not receive any T cells or received 3.times.10.sup.6
cells mock-treated T cells from the same donor. Mice were monitored
for tumor burden and survival for 139 days post administration. The
number of circulating CAR+ T cells was monitored for 28 days post
administration.
[1211] FIG. 19A (high dose) and FIG. 19B (low dose) depict mean
tumor volume of all treated mice. Results from all individual mice
are shown in FIG. 19C (high dose) or FIG. 19D (low dose). As shown
in FIGS. 19A and 19C, JeKo-1 MCL xenograft mice administered
3.times.10.sup.6 (high dose) anti-ROR1 CAR-F expressing cells or
the anti-CD19 CAR+ cells resulted in mean tumor regression (below
the baseline) for at least 111 days (the last day of BLI
measurement). In comparison, mice administered the R12 CAR+ cells
exhibited mean tumor regression for only up to 27 days. As shown in
FIGS. 19B and 19D, at the lower dose of 1.times.10.sup.6 cells,
administration of cells expressing CAR-F resulted in mean tumor
regression for the entire course of the study (up to 111 days). In
comparison, mice administered the anti-CD19 CAR+ cells exhibited
mean tumor regression for up to 36 days, and no mean tumor
regression was observed for mice administered the R12 reference
anti-ROR1 CAR.
[1212] Mice were monitored for body condition at least twice weekly
for 139 days post CAR T cell administration and were euthanized
when moribund or exhibiting signs of severe xenogeneic graft versus
host disease (xGvHD). Kaplan-Meier survival curves of each of the
groups are shown in FIG. 20A (high dose) and FIG. 20B (low dose).
Untreated JeKo-1 xenograft mice had a median survival of 26.5 days.
Animals administered 3.times.10.sup.6 mock-treated cells had a
median survival of 56 days. Median survival was unable to be
determined for all animals treated with either dose of anti-ROR1
CAR-F expressing T cells, as 75% (6 of 8) of animals administered
either the high dose (3.times.10.sup.6 cells; FIG. 20A) or the low
dose (1.times.10.sup.6 cells; FIG. 20B) remained alive at the end
of the study. Notably, the deaths observed in the mice administered
the anti-ROR1 CAR-F were associated with xGvHD rather than lymphoma
burden. Median survival was 70 and 106 days for animals
administered R12 CAR+ T cells at the low and high doses,
respectively. Median survival was unable to be determined for mice
receiving anti-CD19 CAR+ T cells at the low dose (7 of 8 animals
survived to study end) and the median survival was 139 days at the
high dose (4 of 8 animals survived). The deaths in this group was
also primarily associated with xGvHD rather than lymphoma
burden.
[1213] The average number of CAR+ T cells in the peripheral blood
of each mouse was assessed by flow cytometry at days 7, 14, 21 and
28 after CAR+ T cell administration. As shown in FIG. 21, Anti-ROR1
CAR-F expressing cells showed the greatest concentration of
circulating CAR+ T cells in the peripheral blood at day 21 or day
28 after administration at both dose levels examined. At the high
dose, the anti-CD19 CAR+ T cells demonstrated substantially greater
expansion (172.5 cells/.mu.l blood) at day 14 post-administration
than either the anti-ROR1 CAR-F (77.0 cells/.mu.l blood) or R12 CAR
(25.0 cells/.mu.l blood) expressing cells. However, by day 21,
anti-ROR1 CAR-F expressing cells had substantially increased in
counts (140.9 cells/.mu.l blood) compared to both R12 CAR+ (0.3
cells/.mu.l blood) and the anti-CD19 CAR+ (17.2 cells/.mu.l blood)
cells at the low dose. Similar increased expansion was observed at
the high dose anti-ROR1 CAR-F expressing cells, with 392.7
cells/.mu.l blood compared to 10.2 and 66.8 cells/.mu.l blood in
the R12 CAR+ and anti-CD19 CAR+ cells, respectively. This
improvement in expansion was also observed at day 28
post-administration of the cells.
[1214] The results supported high anti-tumor activity, improved T
cell expansion, and prolonged survival of mice administered the
exemplary anti-ROR1 CAR-F in the mouse xenograft model with MCL. By
most measurements, the anti-ROR1 CAR-F-expressing cells showed
substantial improvements compared to the anti-ROR1 reference R12
CAR, and showed similar or improved activity against MCL compared
to cells expressing an anti-CD19 CAR.
Example 7: Epitope Mapping
[1215] Epitopes recognized by the exemplary anti-ROR1
antigen-binding domain contained in the exemplary anti-ROR1 CAR-F
and CAR-G (scFv sequence set forth in SEQ ID NO:118), were assessed
using full discontinuous epitope mapping by Chemical Linkage of
Peptides onto Scaffolds (CLIPS; Pepscan Presto BV, Lelystad, The
Netherlands; see, e.g., Timmerman et al., (2007) J. Mol. Recognit.
20: 283-329). Mapping was carried out using anti-ROR1 scFv clones
in which the scFv was fused with a human Fc (scFv-hFc; IgG1) or a
mouse Fc (scFv-mFc).
[1216] The exemplary anti-ROR1 scFv-mFc and scFv-hFc were expressed
and purified from transiently transfected HEK293 cells, and
assessed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and analytical size exclusion
chromatography (SEC). The extracellular domain of ROR1 (ROR1 ECD)
was recombinantly expressed and purified from transiently
transfected HEK293 cells, and assessed by SDS-PAGE and SEC. The
results showed a highly pure composition of anti-ROR1 scFv-hFc,
scFv-mFc and ROR1-ECD (>99% pure, <1 high molecular weight
multimers for scFv-hFc and ROR1-ECD; >97% pure, <3% high
molecular weight multimers for scFv-mFc).
[1217] Linear and structured synthetic peptide libraries of various
length were generated from human ROR1 (e.g., set forth in SEQ ID
NO:144-146), each offset by one amino acid residue. The structured
peptide library was prepared to fix peptides of different lengths
into defined 3D structures, including any single loop, double loop,
.alpha.-helical and .beta.-turn segment of ROR1. The libraries were
arranged into combinatory maps to yield combinations of loops and
linear peptides representing conformational and/or discontinuous
epitope of ROR1 in a non-biased manner
[1218] An ELISA based heat map was generated on the individual and
combined linear and structured peptides of ROR1 with the binders.
Iterative double alanine substitution of all residues within
peptides that showed positive binding were performed (for wild type
alanine residues, glycine was substituted) Amino acids that
resulted in a 70% or greater reduction in binding when substituted
with alanine were determined to be core components of the
epitope.
[1219] Analysis of epitope mapping showed that an scFv-hFC of the
exemplary anti-ROR1 antigen-binding domain contained in the
exemplary anti-ROR1 CAR-F and CAR-G (scFv sequence set forth in SEQ
ID NO:118) bound to a discontinuous, conformation dependent epitope
primarily centered around amino acids FRSTIYGSRLRIRNL (set forth in
SEQ ID NO:199; corresponding to residues 78-92 of the human ROR1
sequence set forth in SEQ ID NO:144) Table E2 sets forth the
epitopes identified from the epitope mapping. Most of the binding
observed was to combinations with parts of FRSTIYGSRLRIRNL (SEQ ID
NO:199), consistent with some cooperativity between different
regions of the epitope.
TABLE-US-00005 TABLE E2 Binding epitopes of exemplary anti-ROR1
antigen- binding domain Amino acid residue (with reference to SEQ
ID SEQ ID NO: 144) Sequence NO: 5-17 LSVSAELVPTSSW 200 49-62
HCKVSGNPPPTIRW 201 62-87 WFKNDAPVVQEPRRLSFRSTIYGSRL* 202 111-119
VSSTGVLFV 203 117-129 LFVKFGPPPTASP 204 133-143 DEYEEDGFCQP 205
142-155 QPYRGIACARFIGN* 206 191-204 SQFAIPSLCHYAFP 207 202-213
AFPYCDETSSVP 208 228-247 NVLCQTEYIFARSNPMILMR* 209 248-259
LKLPNCEDLPQP 210 259-273 PESPEAANCIRIGIP 211 290-303 VDYRGTVSVTKSGR
212 310-323 SQYPHTHTFTALRF 213 366-380 DSKEKNKMEILYILV 214 Core
residues, determined by .gtoreq.70% reduction in binding upon
double alanine substitution, are underlined and in bold. *likely
contains multiple smaller parts of the epitope.
Example 8: Affinity and Binding Kinetics of Anti-ROR1
Antigen-Binding Domains
[1220] The binding affinity and kinetics of the exemplary anti-ROR1
antigen-binding domains to the antigen ROR1 was assessed by surface
plasmon resonance (SPR).
[1221] A. Binding Affinity and Kinetics
[1222] Equilibrium dissociation constant (K.sub.D), the association
rate constant (k.sub.d or k.sub.on), and dissociation rate constant
(k.sub.d or k.sub.off) of the interaction between recombinant human
ROR1 and the binding domain of exemplary ROR1 CARs was determined
by surface plasmon resonance (SPR) using the Biacore.TM. T200
instrument and Biacore.TM. T200 Evaluation software v3.
Specifically, the scFv binding domain of CARs as described in
Example 3 and Table E1 above, including scFv ROR1-1, ROR1-2, ROR1-3
and ROR1-4 (set forth in SEQ ID NOS: 118, 127, 109 and 134,
respectively; the scFv binding domains of CAR-A, CAR-F, CAR-G,
CAR-I, CAR-R and CAR-B1) and the antigen binding domain of the
reference CAR R12 (SEQ ID NO: 142), were assessed.
[1223] Recombinantly generated anti-ROR1 scFv binding domains and
ROR1 extracellular domain with a C-terminal 6.times.His tag (ROR1
ECD 6.times.His) were used for binding affinity and kinetics
analyses. The binding domains were constructed as a fusion of the
scFv with the Fc portion of murine immunoglobulin heavy chain IgG2a
(scFv-mFc). The murine IgG2a domain contained 4 point mutations to
diminish both Fc receptor and complement binding. Active
concentration of ROR1 ECD 6.times.His was determined by a
calibration-free concentration analysis (CFCA) assay.
[1224] For SPR, an anti-mouse IgG capture surface chip (Mouse
Antibody Capture Kit type 2, catalog number 29215281 and Series S
Sensor Chip CM5, catalog number 29104988, GE Healthcare
Bio-Sciences) was used to capture the anti-ROR1 scFv-mFc binding
domains on a flow cell while another flow cell was left blank for
reference. Multi-cycle kinetics were implemented by subsequent
injections of recombinant human ROR1 at concentrations of 183,
61.0, 20.33, 6.78, and 2.26 nM with variable dissociation over both
the active and reference flow cells.
[1225] ROR1 ECD was injected over the scFv-mFc bound chip at
multiple concentrations using multicycle kinetics. A monovalent 1:1
model was used to analyze the sensograms and to calculate affinity
and kinetic measurements of the interaction.
[1226] FIGS. 22A-22E depict the doubled-reference sensograms for
anti-ROR1 scFv ROR1-1 (SEQ ID NO: 118; FIG. 22A), ROR1-2 (SEQ ID
NO: 127; FIG. 22B), ROR1-3 (SEQ ID NO: 109; FIG. 22C) and ROR1-4
(SEQ ID NO: 134; FIG. 22D) and the scFv antigen binding domain of
the reference CAR R12 (SEQ ID NO: 142; FIG. 22E). The affinity
(equilibrium dissociation constant, K.sub.D) of the interaction
between the anti-ROR1 scFv binding domains and ROR1 are set forth
in Table E3. The affinity (equilibrium dissociation constant,
K.sub.D) of the interaction between the anti-ROR1 scFv ROR1-1
(binding domain of CAR-F) and ROR1 was measured to be 42 nM, and
the kinetic association rate constant (k.sub.a or k.sub.on;
on-rate) and dissociation rate constant (k.sub.d or k.sub.off;
off-rate) of the interaction were observed to be
k.sub.a=1.2.times.10.sup.5 1/Ms and k.sub.d=5.0.times.10.sup.3 1/s,
respectively. The anti-ROR1 scFv ROR1-1 (binding domain of CAR-F)
exhibited substantially higher K.sub.D (lower affinity) and faster
off rate (k.sub.d or k.sub.off) compared to the reference R12 scFv
binding domain The anti-ROR1 scFv ROR1-2 (binding domain of CAR-A)
exhibited substantially lower K.sub.D (higher affinity) and slower
off rate (k.sub.d or k.sub.off) compared to the reference R12 scFv
binding domain
TABLE-US-00006 TABLE E3 Affinity and binding kinetics of exemplary
anti-ROR1 binding domains. scFv # R12 ROR1-1 ROR1-2 ROR1-3 ROR1-4
SEQ ID 142 118 127 134 109 NO: CAR R12 CAR-F, CAR-A, CAR-B1 CAR-I
CAR-G CAR-R K.sub.D (M) 9.420E-10 4.20E-08 9.57E-11 1.38E-08
9.24E-10 k.sub.a or k.sub.on 6.14E+05 1.19E+05 4.12E+05 1.60E+04
1.17E+04 (1/Ms) k.sub.d or k.sub.off 5.79E-04 5.00E-03 3.94E-05
2.20E-04 1.08E-05 (1/s)
[1227] B. Epitope Binning
[1228] SPR was used in epitope binning analysis to compare the
epitope binding regions recognized by the exemplary ROR1 binding
domains compared to the epitope of the anti-ROR1 reference R12
binding domain. Reference R12 scFv binding domain was immobilized
on the surface, ROR1 ECD 6.times.His was introduced and bound to
the R12 binding domain, and recombinant scFv binding domains of
exemplary anti-ROR1 CARs, including scFv ROR1-1, ROR1-2, ROR1-3 and
ROR1-4 (set forth in SEQ ID NOS: 118, 127, 109 and 134,
respectively; the scFv binding domains of CAR-A, CAR-F, CAR-G,
CAR-I, CAR-R and CAR-B1) were injected to assess whether the
recombinant scFv bound to similar regions of ROR1 as the R12
binding domain. The results showed that binding of the scFv R12 to
the ROR1 ECD was competed for by the candidate anti-ROR1 scFv
binding domains ROR1-1, ROR1-2, ROR1-3 and ROR1-4, consistent with
an observation that the candidate scFv binding domains may bind to
an overlapping epitope or region as that bound by the R12 binding
domain
Example 9: Antigen Sensitivity Assessment of Anti-ROR1
Antigen-Binding Domains
[1229] The antigen sensitivity of T cells expressing the exemplary
anti-ROR1 CAR-F and the reference anti-ROR1 CAR R12 were evaluated
for ability to produce cytokines and cytotoxic activity after
stimulation using a ROR1-expressing cell line with regulatable ROR1
expression levels.
[1230] K562 human myelogenous leukemia cell line was engineered to
express ROR1 in response to the tetracycline derivative,
doxycycline (TetOn). As compared to the endogenously ROR1
expressing cell lines, the K562-ROR1-TetOn engineered cell line
expressed very low levels of ROR1 at baseline, even in the absence
of doxycycline treatment.
[1231] 2.times.10.sup.-4 K562-ROR1-TetOn or parental K562 target
cells were transduced to stably express IncuCyte.RTM. NucLight Red.
R10 culture media (RPMI 1640; 1.times.BME; 1.times.NEAA,
1.times.NaPyr; 10% heat inactivated FBS) were added to an optically
clear 96-well flat-bottom plate. Thawed CAR-expressing T cells were
re-suspended at a concentration of 1.6.times.10.sup.6 CAR+ cells/mL
in R10 media. CAR-T were then added to K562 target cells for a
final CAR.sup.+ T cell count of 8.times.10.sup.-4 cells per well
resulting in a 4:1 E:T ratio. Doxycycline hydrochloride was diluted
to the desired concentrations (2 ng/mL-512 ng/mL) using DMSO and
added to the plated CAR-T/target cell mixtures in technical
triplicates. Plates were incubated at 37.degree. C./5% CO.sub.2 for
72 hours with periodic measurement of NucLight Red fluorescence
using the IncuCyte.RTM..
[1232] As shown in FIG. 23, ROR1 expression on K562-ROR1-TetOn
cells after incubation with doxycycline alone showed that ROR1
expression increased with increasing doxycycline concentration.
After 72 hours of incubation, supernatants were collected from
wells containing T cells expressing anti-ROR1 CAR-F or the
reference R12 CAR that had been were mixed with K562-ROR1-TetOn
target cells expressing increasing amounts of ROR1. As shown in
FIG. 24, CAR-F expressing T cells resulted in equivalent or greater
production of IFN.gamma., IL-2 and TNF.alpha. compared to R12
expressing cells, at all ROR1 expression levels tested. As shown in
FIGS. 25A-25B, analysis of cytotoxic function, as determined by
loss of red fluorescent signal over time, showed that CAR-F
expressing cells responded to lower ROR1 levels more effectively
compared to R12 expressing cells, indicating greater antigenic
sensitivity without the loss of specificity as demonstrated by the
lack of response to the parental cell line.
Example 10: Binding Specificity Assessment
[1233] Potential off-target binding and species cross-reactivity of
the binding domain of the exemplary anti-ROR1 CAR-F was assessed by
various methods.
[1234] A. Plasma Membrane Protein Array
[1235] Off-target binding was assessed based on a plasma membrane
protein array. This chip-based platform contains over 4400 groups
of cells genetically engineered to overexpress individual human
extracellular membrane proteins that together represent over 75% of
the human extracellular proteome. The panel also includes over 1000
tethered secreted protein targets. An scFv-Fc (scFv sequence set
forth in SEQ ID NO:118) was generated and used for
immunohistochemistry (IHC) against the cells in the array.
Confirmation screening was performed. The scFv was determined to be
highly selective for ROR1, with no additional specific interactions
identified, including to ROR2. These results indicate low risk for
off-target activity of the anti-ROR1 CAR-F expressing cells
[1236] B. Species Cross-Reactivity
[1237] Table E4 shows the extracellular domain (ECD) homology for
ROR1 between human (Homo sapiens), Rhesus macaques (Macaca
mulatta), cynomolgus macaques (Macaca fasicularis), and mice (Mus
musculus). ROR1 is 100% conserved in the ECD between human and
non-human primates.
TABLE-US-00007 TABLE E4 ROR1 ECD Homology Across Species Macaca
mulatta Macaca fasicularis Mus musculus Homo sapiens 100% 100%
98.7% Extracellular domain a.a. 30-406 of human ROR1 Homo sapiens:
Uniprot Q01973 (set forth in SEQ ID NO: 214) Macaca mulatta:
Uniprot F6RUP2 (set forth in SEQ ID NO: 215) Macaca fasicularis:
Uniprot A0A2K5WTX7 (set forth in SEQ ID NO: 216), Uniprot
A0A2K5WTX4 (set forth in SEQ ID NO: 217) Mus musculus: Uniprot
Q9Z139 (set forth in SEQ ID NO: 218)
[1238] Species cross-reactivity of anti-ROR1 CAR-F and R12 CAR was
assessed by co-culturing an engineered Jurkat T cell line
containing a Nur77-tdTomato reporter and expressing the anti-ROR1
CAR-F or the reference R12 CAR (as described above in Example 1.C),
with CT26 murine fibroblasts engineered to express a mouse ROR1
(CT26-mROR1), CT26 cells engineered to express a human ROR1
(CT26-hROR1), or unmodified CT26 cells or unmodified K562 human
myelogenous leukemia cells, and assessing expression of the
reporter.
[1239] As shown in FIG. 26, although there is 98.7% homology
between human and mouse ROR1, no cross-reactivity was observed for
binding of the anti-ROR1 CAR-F or R12 CAR to mouse ROR1-expressing
target cells.
[1240] The present invention is not intended to be limited in scope
to the particular disclosed embodiments, which are provided, for
example, to illustrate various aspects of the invention. Various
modifications to the compositions and methods described will become
apparent from the description and teachings herein. Such variations
may be practiced without departing from the true scope and spirit
of the disclosure and are intended to fall within the scope of the
present disclosure.
TABLE-US-00008 Sequences SEQ ID NO: Sequence Description 1
ESKYGPPCPPCP Spacer (IgG4 hinge) (aa) 2
gaatctaagtacggaccgccctgccccccttgccct Spacer (IgG4 hinge)(nt) 3
ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
Hinge-C.sub.H3
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS spacer
(aa) LGK 4
GAATCTAAGTACGGACCGCCTTGTCCTCCATGTCCTGGCCAGCCAAGAGAACCCCAGG
Hinge-C.sub.H3
TGTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTG spacer
(nt) CCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAG
CCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCC
TGTACAGCCGGCTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTG
CAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGAGC
CTGGGCAAG 5
RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERET
IgD-hinge-Fc
KTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGG
VEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAP
VKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPG
STTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH 6
LEGGGEGRGSLLTCGDVEENPGPR T2A 7
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDL tEGFR
HILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIR
GRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSG
QKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE
GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMG
ENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLV
VALGIGLFM 8 FWVLVVVGGVLACYSLLVTVAFIIFWV CD28 (amino acids 153-179
of Accession No. P10747) 9
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVT CD28
(amino VAFIIFWV acids 114-179 of Accession No. P10747) 10
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (amino acids 180-220
of P10747) 11 RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (LL to
GG) 12 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1 BB (amino
acids 214-255 of Q07011.1) 13
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL CD3 zeta
YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 14
RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL CD3 zeta
YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 15
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL CD3 zeta
YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 16
RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQE tEGFR
LDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLG
LRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCH
ALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQ
AMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPN
CTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM 17 EGRGSLLTCGDVEENPGP
T2A 18 GSGATNFSLLKQAGDVEENPGP P2A 19 ATNFSLLKQAGDVEENPGP P2A 20
QCTNYALLKLAGDVESNPGP E2A 21 VKQTLNFDLLKLAGDVESNPGP F2A 22
PGGG-(SGGGG)5-P- wherein P is proline, G is glycine and S linker is
serine 23 GSADDAKKDAAKKDGKS Linker 24 GSTSGSGKPGSGEGSTKG Linker 25
X.sub.1PPX.sub.2P X1 is glycine, cysteine or arginine X2 is
cysteine Hinge or threonine 26 EPKSCDKTHTCPPCP Hinge 27
ERKCCVECPPCP Hinge 28
ELKTPLGDTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCP Hinge
RCP 29 ESKYGPPCPSCP Hinge 30 gagtctaaatacggaccgccttgtcctccttgtccc
Spacer (IgG4hinge) (nt) O/SSE 31 YGPPCPPCP Hinge 32 KYGPPCPPCP
Hinge 33 EVVVKYGPPCPPCP Hinge 34
ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW
Hinge-C.sub.H2-
YVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT C.sub.H3
spacer ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
aa TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 35
GAATCTAAGTACGGACCGCCTTGTCCTCCATGTCCTGCTCCTCCAGTTGCCGGACCTT
Hinge-C.sub.H2-
CCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGA C.sub.H3
spacer AGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAACTGG
(nt TATGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCC
AGAGCACCTACAGAGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGG
CAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACC
ATCAGCAAGGCCAAGGGCCAGCCAAGAGAACCCCAGGTGTACACACTGCCTCCAAGCC
AAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCC
TTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACC
ACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCCGGCTGACCGTGG
ACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCT
GCACAACCACTACACCCAGAAGTCTCTGAGCCTGAGCCTGGGCAAG 36
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN
Hinge-C.sub.H2-
WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK C.sub.H3
spacer TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
(aa) TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 37
ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW
IgG4/IgG2 YV hinge-
DGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS
IgG2/IgG4 KA C.sub.H2-IgG4
KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV C.sub.H3
spacer LD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (aa) 38
gaatctaagtacggaccgccctgccctccctgccctgctcctcctgtggctggaccaa
IgG4/IgG2
gcgtgttcctgtttccacctaagcctaaagataccctgatgatttcccgcacacctga hinge-
agtgacttgcgtggtcgtggacgtgagccaggaggatccagaagtgcagttcaactgg
IgG2/IgG4
tacgtggacggcgtggaagtccacaatgctaagactaaaccccgagaggaacagtttc
C.sub.H2- IgG4
agtcaacttaccgggtcgtgagcgtgctgaccgtcctgcatcaggattggctgaacgg C.sub.H3
spacer gaaggagtataagtgcaaagtgtctaataagggactgcctagctccatcgagaaaaca
(nt) attagtaaggcaaaagggcagcctcgagaaccacaggtgtataccctgccccctagcc
aggaggaaatgaccaagaaccaggtgtccctgacatgtctggtcaaaggcttctatcc
aagtgacatcgccgtggagtgggaatcaaatgggcagcccgagaacaattacaagacc
acaccacccgtgctggactctgatggaagtttctttctgtattccaggctgaccgtgg
ataaatctcgctggcaggagggcaacgtgttctcttgcagtgtcatgcacgaagccct
gcacaatcattatacacagaagtcactgagcctgtccctgggcaaa 39 GGGGS 4GS linker
(aa) 40 GGGS 3GS linker (aa) 41 GGGGSGGGGSGGGGS (4GS).sub.3 linker
(aa) 42 MPLLLLLPLLWAGALA CD33 Signal Peptide 43
MVLQTQVFISLLLWISGAYG human IgG- kappa signal peptide (aa) 44
atggtgctgcagacccaggtgttcatcagcctgctgctgtggatctccggagcatacg human
IgG- ga kappa signal sequence (nt) 45 MLLLVTSLLLCELPHPAFLLIP GMCSFR
alpha chain signal peptide (aa) 46
atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcc GMCSFR
tgatccca alpha chain signal sequence (nt) 47 MALPVTALLLPLALLLHA CD8
alpha signal peptide 48
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ Human
IgG2 SSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAG Fc
(Uniprot PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
P01859) FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 49
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ Human
IgG4 SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLG Fc
(Uniprot GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE
P01861) QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP
PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL
TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 50 GYTFTSY CDR-H1 51
GYTFTSYGIS CDR-H1 52 SYGIS CDR-H1 53 GYTFTSYG CDR-H1 54 SAYNGN
CDR-H2 55 WISAYNGNTK CDR-H2 56 WISAYNGNTKYAQKLQG CDR-H2 57 ISAYNGNT
CDR-H2 58 DEDILTGYNYYGMDV CDR-H3 59 ARDEDILTGYNYYGMDV CDR-H3 60
TLSSGHSSYAILA CDR-L1 61 SGHSSYA, CDR-L1 62 LNSDGSHSKGD CDR-L2 63
LNSDGSH CDR-L2 64 QTWGTGIRV CDR-L3 65 GGSISNY CDR-H1
66 GGSISNYYWS CDR-H1 67 NYYWS CDR-H1 68 GGSISNYY CDR-H1 69 YTSGS
CDR-H2 70 RIYTSGSTN CDR-H2 71 RIYTSGSTNYNPSLKS CDR-H2 72 IYTSGST
CDR-H2 73 YYDILTGFFDY CDR-H3 74 ARYYDILTGFFDY CDR-H3 75 RMSQDISSYLA
CDR-L1 76 QDISSY CDR-L1 77 AASSLQS CDR-L2 78 AAS CDR-L2 79
QQYDSFPPT CDR-L3 80 GGSINSTTS CDR-H1 81 GGSINSTTSYWA CDR-H1 82
STTSYWA CDR-H1 83 GGSINSTTSY CDR-H1 84 FYSGK CDR-H2 85 TIFYSGKTY
CDR-H2 86 TIFYSGKTYNNPSLKS CDR-H2 87 IFYSGKT CDR-H2 88 FDYGFHDAFDI
CDR-H3 89 ARFDYGFHDAFDI CDR-H3 90 RASQSITSDYLS CDR-L1 91 QSITSDY
CDR-L1 92 GASTRAT CDR-L2 93 GAS CDR-L2 94 QQDYNLTYT CDR-L3 95
SAYTGN CDR-H2 96 WISAYTGNTR CDR-H2 97 WISAYTGNTRYAQKLOG CDR-H2 98
ISAYTGNT CDR-H2 99 EEGATTDYDYYGMDV CDR-H3 100 AREEGATTDYDYYGMDV
CDR-H3 101
CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGG V.sub.H
(nt) TCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACA
GGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACA
AAGTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCA
CAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGC
GAGAGATGAGGATATTTTGACTGGTTACAACTACTACGGTATGGACGTCTGGGGCCAA
GGGACCACGGTCACCGTCTCCTCA 102
caggtgcagctggttcaatctggcgccgaagtgaagaaaccaggcgcctctgtgaagg V.sub.H
(nt) tgtcctgcaaggccagcggctacacctttaccagctacggcatcagctgggtccgaca
O/SSE ggctcctggacaaggcttggaatggatgggctggatcagcgcctacaacggcaacacc
aaatacgcccagaaactgcagggcagagtgaccatgaccaccgacaccagcacaagca
ccgcctacatggaactgcggagcctgagatccgatgacaccgccgtgtactactgcgc
cagagatgaggacatcctgaccggctacaactactacggcatggacgtgtggggccag
ggcacaacagtgacagtttcttct 103
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNT V.sub.H
(aa) KYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDEDILTGYNYYGMDVWGQ
GTTVTVSS 104
CAGCTTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAGC V.sub.L
(nt) TCACCTGCACTCTGAGCAGTGGGCACAGCAGCTACGCCATCGCATGGCATCAGCAGCA
GCCAGAGAAGGGCCCTCGGTACTTGATGAAGCTTAACAGTGATGGCAGCCACAGCAAG
GGGGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGCTACCTCA
CCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGACCTGGGGCAC
TGGCATTCGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGC 105
caactggtgctgacacagtctcctagcgcctctgcttctctgggagccagcgtgaagc V.sub.L
(nt) tgacctgtacactgtctagcggccacagcagctacgccattgcttggcatcagcagca
O/SSE gcccgagaagggccctagatacctgatgaagctgaacagcgacggcagccactctaaa
ggcgacggcatccccgatagattcagcggcagttctagcggagccgagcgctacctga
caatcagctctctgcaatccgaggacgaggccgactactactgtcagacatggggcac
cggcatcagagtgtttggcggaggcaccaagctgacagtgcttgga 106
QLVLTQSPSASASLGASVKLTCTLSSGHSSYAIAWHQQQPEKGPRYLMKLNSDGSHSK V.sub.L
(aa) GDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCQTWGTGIRVFGGGTKLTVLG 107
CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGG scFv
(V.sub.H-V.sub.L)
TCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACA (nt)
GGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACA
AAGTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCA
CAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGC
GAGAGATGAGGATATTTTGACTGGTTACAACTACTACGGTATGGACGTCTGGGGCCAA
GGGACCACGGTCACCGTCTCCTCAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTG
GTGGTGGATCCCAGCTTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGC
CTCGGTCAAGCTCACCTGCACTCTGAGCAGTGGGCACAGCAGCTACGCCATCGCATGG
CATCAGCAGCAGCCAGAGAAGGGCCCTCGGTACTTGATGAAGCTTAACAGTGATGGCA
GCCACAGCAAGGGGGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGA
GCGCTACCTCACCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAG
ACCTGGGGCACTGGCATTCGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGC 108
caggtgcagctggttcaatctggcgccgaagtgaagaaaccaggcgcctctgtgaagg scFv
(V.sub.H-V.sub.L)
tgtcctgcaaggccagcggctacacctttaccagctacggcatcagctgggtccgaca (nt)
O/SSE ggctcctggacaaggcttggaatggatgggctggatcagcgcctacaacggcaacacc
aaatacgcccagaaactgcagggcagagtgaccatgaccaccgacaccagcacaagca
ccgcctacatggaactgcggagcctgagatccgatgacaccgccgtgtactactgcgc
cagagatgaggacatcctgaccggctacaactactacggcatggacgtgtggggccag
ggcacaacagtgacagtttcttctggcggcggaggatctggcggaggtggaagcggag
gcggtggatctcaactggtgctgacacagtctcctagcgcctctgcttctctgggagc
cagcgtgaagctgacctgtacactgtctagcggccacagcagctacgccattgcttgg
catcagcagcagcccgagaagggccctagatacctgatgaagctgaacagcgacggca
gccactctaaaggcgacggcatccccgatagattcagcggcagttctagcggagccga
gcgctacctgacaatcagctctctgcaatccgaggacgaggccgactactactgtcag
acatggggcaccggcatcagagtgtttggcggaggcaccaagctgacagtgcttgga 109
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNT scFv
(V.sub.H-V.sub.L)
KYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDEDILTGYNYYGMDVWGQ (aa)
GTTVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSGHSSYAIAW
HQQQPEKGPRYLMKLNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCQ
TWGTGIRVFGGGTKLTVLG 110
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCC V.sub.H
(nt) TCACCTGCACTGTCTCTGGAGGCTCCATCAGTAATTACTACTGGAGCTGGATCCGGCA
GCCCGCCGGGAAGGGACTGGAGTGGATTGGGCGTATCTATACCAGTGGGAGCACCAAC
TACAACCCCTCCCTCAAGAGTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAGT
TCTCCCTGAAGCTGAGTTCTTTGACCGCCGCGGACACGGCCATATATTACTGTGCGAG
GTATTACGATATTTTGACTGGTTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACC
GTCTCCTCA 111
Caggttcagctgcaagagtctggccctggcctggtcaagcctagcgaaacactgagcc V.sub.H
(nt) tgacctgtaccgtgtctggcggcagcatctccaactactactggtcctggatcagaca
O/SSE gcctgccggcaaaggcctggaatggatcggcagaatctacaccagcggcagcaccaac
tacaaccccagcctgaagtccagagtgaccatgagcgtggacaccagcaagaaccagt
tctccctgaagctgagcagcctgacagccgccgataccgccatctactactgtgcccg
gtactacgatatcctgaccggcttcttcgactactggggccagggaacactggtcaca
gtttctagc 112
QVQLQESGPGLVKPSETLSLTCTVSGGSISNYYWSWIRQPAGKGLEWIGRIYTSGSTN V.sub.H
(aa) YNPSLKSRVTMSVDTSKNQFSLKLSSLTAADTAIYYCARYYDILTGFFDYWGQGTLVT VSS
113 GTCATCTGGATGACCCAGTCTCCATCCTTACTCTCTGCATCTACAGGAGACAGTGTCA
V.sub.L (nt)
CCATCAGTTGTCGGATGAGTCAGGACATTAGCAGTTATTTAGCCTGGTATCAGCAAAA
ACCAGGGAAAGCCCCTGAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTC
CCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGTTCCC
TGCAGTCTGAAGATTTTGCTACTTATTACTGTCAACAGTATGATAGTTTCCCTCCGAC
GTTCGGCCAAGGGACCAAGGTGGAATTCAAACGG 114
Gtgatttggatgacacagagccctagcctgctgagcgccagcacaggcgatagcgtga VL (nt)
ccatcagctgcagaatgagccaggacatcagcagctacctggcttggtatcagcagaa O/SSE
gcctggcaaggcccctgaactgctgatctatgccgcttccagtctgcagagcggcgtg
ccatctagattttccggcagcggctctggcaccgacttcaccctgacaatcagctccc
tgcagtccgaggacttcgccacctactattgccagcagtacgacagcttccctccaac
ctttggccagggcaccaaggtggaattcaagcgc 115
VIWMTQSPSLLSASTGDSVTISCRMSQDISSYLAWYQQKPGKAPELLIYAASSLQSGV V.sub.L
(aa) PSRFSGSGSGTDFTLTISSLQSEDFATYYCQQYDSFPPTFGQGTKVEFKR 116
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCC scFv
(V.sub.H-V.sub.L)
TCACCTGCACTGTCTCTGGAGGCTCCATCAGTAATTACTACTGGAGCTGGATCCGGCA (nt)
GCCCGCCGGGAAGGGACTGGAGTGGATTGGGCGTATCTATACCAGTGGGAGCACCAAC
TACAACCCCTCCCTCAAGAGTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAGT
TCTCCCTGAAGCTGAGTTCTTTGACCGCCGCGGACACGGCCATATATTACTGTGCGAG
GTATTACGATATTTTGACTGGTTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACC
GTCTCCTCAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCGTCA
TCTGGATGACCCAGTCTCCATCCTTACTCTCTGCATCTACAGGAGACAGTGTCACCAT
CAGTTGTCGGATGAGTCAGGACATTAGCAGTTATTTAGCCTGGTATCAGCAAAAACCA
GGGAAAGCCCCTGAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCAT
CAAGGTTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGTTCCCTGCA
GTCTGAAGATTTTGCTACTTATTACTGTCAACAGTATGATAGTTTCCCTCCGACGTTC
GGCCAAGGGACCAAGGTGGAATTCAAACGG 117
caggttcagctgcaagagtctggccctggcctggtcaagcctagcgaaacactgagcc scFV
(V.sub.H-V.sub.L)
tgacctgtaccgtgtctggcggcagcatctccaactactactggtcctggatcagaca (nt)
O/SSE gcctgccggcaaaggcctggaatggatcggcagaatctacaccagcggcagcaccaac
tacaaccccagcctgaagtccagagtgaccatgagcgtggacaccagcaagaaccagt
tctccctgaagctgagcagcctgacagccgccgataccgccatctactactgtgcccg
gtactacgatatcctgaccggcttcttcgactactggggccagggaacactggtcaca
gtttctagcggaggcggaggatctggtggcggaggaagtggcggaggcggttctgtga
tttggatgacacagagccctagcctgctgagcgccagcacaggcgatagcgtgaccat
cagctgcagaatgagccaggacatcagcagctacctggcttggtatcagcagaagcct
ggcaaggcccctgaactgctgatctatgccgcttccagtctgcagagcggcgtgccat
ctagattttccggcagcggctctggcaccgacttcaccctgacaatcagctccctgca
gtccgaggacttcgccacctactattgccagcagtacgacagcttccctccaaccttt
ggccagggcaccaaggtggaattcaagcgc 118
QVQLQESGPGLVKPSETLSLTCTVSGGSISNYYWSWIRQPAGKGLEWIGRIYTSGSTN sCFV
(V.sub.H-V.sub.L)
YNPSLKSRVTMSVDTSKNQFSLKLSSLTAADTAIYYCARYYDILTGFFDYWGQGTLVT (aa)
VSSGGGGSGGGGSGGGGSVIWMTQSPSLLSASTGDSVTISCRMSQDISSYLAWYQQKP
GKAPELLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYYCQQYDSFPPTF
GQGTKVEFKR 119
CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCC V.sub.H
(nt) TCACCTGCACTGTCTCTGGTGGCTCCATCAACAGTACTACTTCCTACTGGGCCTGGAT
CCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGACTATCTTTTATAGTGGGAAA
ACCTACAACAACCCGTCCCTCAAGAGTCGAGTCACCATGTCCGTAGACACGTCCAAGA
ACCACTTCTCCCTGAAGGTGAACTCTGTGACCGCCGCAGACACGGCTGTGTATTACTG
TGCGAGGTTTGACTACGGTTTTCATGATGCTTTTGATATCTGGGGCCAGGGGACAATG
GTCACCGTCTCTTCA 120
cagctccagctgcaagaatctggacctggcctggtcaagcccagcgagacactgtctc V.sub.H
(nt) tgacctgtacagtgtccggcggcagcatcaatagcaccacaagctactgggcctggat
O/SSE cagacagcctcctggcaaaggcctggaatggatcggcaccatcttctacagcggcaag
acctacaacaaccccagcctgaagtccagagtgaccatgagcgtggacaccagcaaga
accacttcagcctgaaagtgaacagcgtgacagccgccgataccgccgtgtactactg
cgccagattcgactacggcttccacgacgccttcgacatctggggccagggcacaatg
gtcacagtttctagc 121
QLQLQESGPGLVKPSETLSLTCTVSGGSINSTTSYWAWIRQPPGKGLEWIGTIFYSGK V.sub.H
(aa) TYNNPSLKSRVTMSVDTSKNHFSLKVNSVTAADTAVYYCARFDYGFHDAFDIWGQGTM
VTVSS 122
GAAATTGTAATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCA V.sub.L
(nt) CCCTCTCCTGCAGGGCCAGTCAGAGTATTACCAGCGACTACTTATCCTGGTACCAACA
AAAACCTGGGCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGC
ATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCA
GCCTGCAGCCTGAAGATTTTGTAGTTTATTACTGTCAGCAGGATTATAACTTGTACAC
TTTTGGCCAGGGGACCAAGCTGGAGATCAAACGG 123
gagattgtgatgacacagagccccgccactctgagccttagtcctggcgaaagagcca V.sub.L
(nt) cactgagctgcagagccagccagagcatcaccagcgattacctgagctggtatcagca
O/SSE gaagcccggacaggctcccagactgctgatctatggcgcctctacaagagccaccggc
attcccgcccgcttttctggctctggaagcggcaccgacttcaccctgaccatatcta
gcctgcagcctgaggacttcgtggtgtactattgccagcaggactacaacctgtacac
cttcggccaggggaccaagctggaaatcaagaga 124
EIVMTQSPATLSLSPGERATLSCRASQSITSDYLSWYQQKPGQAPRLLIYGASTRATG V.sub.L
(aa) IPARFSGSGSGTDFTLTISSLQPEDFVVYYCQQDYNLYTFGQGTKLEIKR 125
CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCC scFv
(V.sub.H-V.sub.L)
TCACCTGCACTGTCTCTGGTGGCTCCATCAACAGTACTACTTCCTACTGGGCCTGGAT (nt)
CCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGACTATCTTTTATAGTGGGAAA
ACCTACAACAACCCGTCCCTCAAGAGTCGAGTCACCATGTCCGTAGACACGTCCAAGA
ACCACTTCTCCCTGAAGGTGAACTCTGTGACCGCCGCAGACACGGCTGTGTATTACTG
TGCGAGGTTTGACTACGGTTTTCATGATGCTTTTGATATCTGGGGCCAGGGGACAATG
GTCACCGTCTCTTCAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGAT
CCGAAATTGTAATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGC
CACCCTCTCCTGCAGGGCCAGTCAGAGTATTACCAGCGACTACTTATCCTGGTACCAA
CAAAAACCTGGGCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTG
GCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAG
CAGCCTGCAGCCTGAAGATTTTGTAGTTTATTACTGTCAGCAGGATTATAACTTGTAC
ACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGG 126
Cagctccagctgcaagaatctggacctggcctggtcaagcccagcgagacactgtctc scFv
(V.sub.H-V.sub.L)
tgacctgtacagtgtccggcggcagcatcaatagcaccacaagctactgggcctggat (nt)
O/SSE cagacagcctcctggcaaaggcctggaatggatcggcaccatcttctacagcggcaag
acctacaacaaccccagcctgaagtccagagtgaccatgagcgtggacaccagcaaga
accacttcagcctgaaagtgaacagcgtgacagccgccgataccgccgtgtactactg
cgccagattcgactacggcttccacgacgccttcgacatctggggccagggcacaatg
gtcacagtttctagcggaggcggaggatctggtggcggaggaagtggcggaggcggtt
ctgagattgtgatgacacagagccccgccactctgagccttagtcctggcgaaagagc
cacactgagctgcagagccagccagagcatcaccagcgattacctgagctggtatcag
cagaagcccggacaggctcccagactgctgatctatggcgcctctacaagagccaccg
gcattcccgcccgcttttctggctctggaagcggcaccgacttcaccctgaccatatc
tagcctgcagcctgaggacttcgtggtgtactattgccagcaggactacaacctgtac
accttcggccaggggaccaagctggaaatcaagaga 127
QLQLQESGPGLVKPSETLSLTCTVSGGSINSTTSYWAWIRQPPGKGLEWIGTIFYSGK scFv
(V.sub.H-V.sub.L)
TYNNPSLKSRVTMSVDTSKNHFSLKVNSVTAADTAVYYCARFDYGFHDAFDIWGQGTM (aa)
VTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQSITSDYLSWYQ
QKPGQAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISSLQPEDFVVYYCQQDYNLY
TFGQGTKLEIKR 128
CAGGTTCAGCTGCTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGG V.sub.H
(nt) TCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACA
GGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACACTGGTAACACA
AGGTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCA
CAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGC
GAGAGAAGAAGGAGCTACTACGGACTACGACTACTACGGTATGGACGTCTGGGGCCAA
GGGACTGCGGTCACCGTCTCCTCA 129
caggttcagctgcttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaagg V.sub.H
(nt) tgtcctgcaaggccagcggctacacctttaccagctacggcatcagctgggtccgaca
O/SSE ggctcctggacaaggcttggaatggatgggctggatcagcgcctacaccggcaatacc
agatacgcccagaaactgcagggcagagtgaccatgaccaccgacaccagcacaagca
ccgcctacatggaactgcggagcctgagatccgatgacaccgccgtgtactactgcgc
cagagaagaaggcgccaccaccgactacgactactacggcatggatgtgtggggccag
ggaacagccgtgacagtttcttct 130
QVQLLQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYTGNT V.sub.H
(aa) RYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAREEGATTDYDYYGMDVWGQ
GTAVTVSS 131
caactggttctgacacagagcccaagcgcctctgcatctctgggagcttccgtgaagc V.sub.L
(nt) tgacctgcacactgtctagcggccacagcagctatgccattgcctggcatcagcaaca
O/SSE gcccgagaagggccctagatacctgatgaagctgaacagcgacggcagccactctaaa
ggcgacggcatccccgatagattcagcggcagttctagcggagccgagcgctacctga
caatcagctctctgcaatccgaggacgaggccgattactactgtcagacatggggcac
cggcatcagagtgtttggcggcggaacaaagctgaccgtgctgggc 132
CAGGTTCAGCTGCTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGG scFv
(V.sub.H-V.sub.L)
TCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACA (nt)
GGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACACTGGTAACACA
AGGTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCA
CAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGC
GAGAGAAGAAGGAGCTACTACGGACTACGACTACTACGGTATGGACGTCTGGGGCCAA
GGGACTGCGGTCACCGTCTCCTCAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTG
GTGGTGGATCCCAGCTTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGC
CTCGGTCAAGCTCACCTGCACTCTGAGCAGTGGGCACAGCAGCTACGCCATCGCATGG
CATCAGCAGCAGCCAGAGAAGGGCCCTCGGTACTTGATGAAGCTTAACAGTGATGGCA
GCCACAGCAAGGGGGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGA
GCGCTACCTCACCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAG
ACCTGGGGCACTGGCATTCGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGC 133
caggttcagctgcttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaagg scFv
(V.sub.H-V.sub.L)
tgtcctgcaaggccagcggctacacctttaccagctacggcatcagctgggtccgaca (nt)
O/SSE ggctcctggacaaggcttggaatggatgggctggatcagcgcctacaccggcaatacc
agatacgcccagaaactgcagggcagagtgaccatgaccaccgacaccagcacaagca
ccgcctacatggaactgcggagcctgagatccgatgacaccgccgtgtactactgcgc
cagagaagaaggcgccaccaccgactacgactactacggcatggatgtgtggggccag
ggaacagccgtgacagtttcttctggtggcggaggatctggcggaggtggaagcggcg
gaggcggatctcaactggttctgacacagagcccaagcgcctctgcatctctgggagc
ttccgtgaagctgacctgcacactgtctagcggccacagcagctatgccattgcctgg
catcagcaacagcccgagaagggccctagatacctgatgaagctgaacagcgacggca
gccactctaaaggcgacggcatccccgatagattcagcggcagttctagcggagccga
gcgctacctgacaatcagctctctgcaatccgaggacgaggccgattactactgtcag
acatggggcaccggcatcagagtgtttggcggcggaacaaagctgaccgtgctgggc 134
QVQLLQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYTGNT scFv
(V.sub.H-V.sub.L)
RYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAREEGATTDYDYYGMDVWGQ (aa)
GTAVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSGHSSYAIAW
HQQQPEKGPRYLMKLNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCQ
TWGTGIRVFGGGTKLTVLG 135 ESKYGPPCPPCPM IgG4 hinge spacer (aa) 136
gagtctaaatacggaccgccttgtcctccttgtcccatg IgG4 hinge spacer (nt)
O/SSE 137
gagtctaaatacggaccgccttgtcctccttgtcccggccagccaagagagccccagg
Hinge-C.sub.H3
tttacacactgcctccaagccaagaggaaatgaccaagaatcaggtgtccctgacatg spacer
(nt) cctggtcaagggcttctacccctccgatatcgccgtggaatgggagagcaatggccag
O/SSE cctgagaacaactacaagaccacacctcctgtgctggacagcgacggcagtttcttcc
tgtatagtagactcaccgtggataaatcaagatggcaagagggcaacgtgttcagctg
cagcgtgatgcacgaggccctgcacaaccactacacccagaaaagcctgagcctgtct
ctgggcaag 138
ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
Hinge-C.sub.H3
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS spacer
(aa) LGKM 139
gagtctaaatacggaccgccttgtcctccttgtcccggccagccaagagagccccagg
Hinge-C.sub.H3
tttacacactgcctccaagccaagaggaaatgaccaagaatcaggtgtccctgacatg spacer
(nt) cctggtcaagggcttctacccctccgatatcgccgtggaatgggagagcaatggccag
O/SSE cctgagaacaactacaagaccacacctcctgtgctggacagcgacggcagtttcttcc
tgtatagtagactcaccgtggataaatcaagatggcaagagggcaacgtgttcagctg
cagcgtgatgcacgaggccctgcacaaccactacacccagaaaagcctgagcctgtct
ctgggcaagatg 140
gagtctaaatacggaccgccttgtcctccttgtcccgctcctcctgttgccggacctt
IgG4/IgG2
ccgtgttcctgtttcctccaaagcctaaggacaccctgatgatcagcaggacccctga hinge-
agtgacctgcgtggtggtggatgtgtcccaagaggatcccgaggtgcagttcaactgg
IgG2/IgG4
tatgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagttcc
C.sub.H2-IgG4
agagcacctacagagtggtgtccgtgctgacagtgctgcaccaggattggctgaacgg C.sub.H3
spacer caaagagtacaagtgcaaggtgtccaacaagggcctgcctagcagcatcgagaaaacc
(nt) O/SSE
atctccaaggccaagggccagccaagagagccccaggtttacacactgcctccaagcc
aagaggaaatgaccaagaatcaggtgtccctgacatgcctggtcaagggcttctaccc
ctccgatatcgccgtggaatgggagagcaatggccagcctgagaacaactacaagacc
acacctcctgtgctggacagcgacggcagtttcttcctgtatagtagactcaccgtgg
ataaatcaagatggcaagagggcaacgtgttcagctgcagcgtgatgcacgaggccct
gcacaaccactacacccagaaaagcctgagcctgtctctgggcaag 141
cttgaaggtggtggcgaaggcagaggcagcctgcttacatgcggagatgtggaagaga T2A (nt)
accccggacctaga O/SSE 142
QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKT R12
V.sub.H-V.sub.L
YYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTL scFv
(aa) VTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQ
LQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGADY
IGGYVFGGGTQLTVTG 143
caagaacagctggtggaatctggcggcagactggttacacctggcggaagcctgacac R12
V.sub.H-V.sub.L
tgagctgtaaagccagcggcttcgacttcagcgcctactacatgagctgggtccgaca scFv
(nt) ggcccctggcaaaggactggaatggatcgccacaatctaccccagctccggcaagacc
tactacgccacatgggtcaacggccggttcaccatcagcagcgacaacgcccagaaca
ccgtggacctgcagatgaactctctgacagccgccgaccgggccacctacttttgtgc
cagagatagctacgccgacgacggcgccctgttcaatatttggggacctggcacactc
gtgaccatctctagcggaggcggaggaagtggtggcggaggatcaggcggtggtggat
ctgaactggtgctgacacagagcccctctgtgtctgctgctctgggaagccctgccaa
gatcacatgtaccctgagcagcgcccacaagaccgacaccatcgactggtatcagcag
ctgcagggcgaagcccctagatacctgatgcaggttcagagcgacggcagctacacca
aaagacctggcgtgcccgatagattcagcggcagttcttctggcgccgatcgctacct
gatcatcccttctgtgcaagccgacgatgaggccgactattactgcggagccgattac
atcggcggctacgttttcggtggcggcacacagttgacagtgacaggcg 144
QETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPP human
ROR1 TIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGV
(aa) LFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQIT
GenBank: AAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQ
AAA60275.1
TEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGV
DYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTL
DENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSS
SAPVQRQPKHVRGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLY
LPGMDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCML
FEYINQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSH
FFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSLLPIRWMPPEAIMYG
KFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSEDCPPRMYSLM
TECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLSN
PRYPNYMFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFPAAHYQPTGP
PRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGGMGI
TVFGNKSQKPYKIDSKQASLLGDANIHGHTESMISAEL 145
QETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPP human
ROR1 TIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGV
isoform 2 (aa)
LFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQIT
AAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQ
TEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGV
DYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTL
DENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSS
SAPVQRQPKHVRGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLY
LPGMDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVC 146
QETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPP human
ROR1 TIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGV
isoform 3 (aa)
LFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQIT
AAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQ
TEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGV
DYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTL
DENFKSDLCDIPACGK 147
atgttttgggtgctggtcgtggtcggaggggtgctggcctgttacagcctgctggtga CD28
cagtcgctttcatcatcttctgggtg transmembrane domain (nt) 148
atgttctgggtgctcgtggtcgttggcggagtgctggcctgttacagcctgctggtta CD28
ccgtggccttcatcatcttttgggtc transmembrane domain (nt) (O/SSE) 149
MFWVLVVVGGVLACYSLLVTVAFIIFWV CD28 transmembrane domain (aa) 150
agagtgaagttcagcagatccgccgacgctccagcctatcagcagggccaaaaccagc CD3-zeta
tgtacaacgagctgaacctggggagaagagaagagtacgacgtgctggataagcggag derived
aggcagagatcctgaaatgggcggcaagcccagacggaagaatcctcaagagggcctg
intracellular
tataatgagctgcagaaagacaagatggccgaggcctacagcgagatcggaatgaagg
signaling
gcgagcgcagaagaggcaagggacacgatggactgtaccagggcctgagcaccgccac domain
(nt)
caaggatacctatgacgcactgcacatgcaggccctgccacctaga (O/SSE) 151
atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcc
truncated
tgatcccacgcaaagtgtgtaacggaataggtattggtgaatttaaagactcactctc EGFR
cataaatgctacgaatattaaacacttcaaaaactgcacctccatcagtggcgatctc (tEGFR)
cacatcctgccggtggcatttaggggtgactccttcacacatactcctcctctggatc sequence
(nt) cacaggaactggatattctgaaaaccgtaaaggaaatcacagggtttttgctgattca
ggcttggcctgaaaacaggacggacctccatgcctttgagaacctagaaatcatacgc
ggcaggaccaagcaacatggtcagttttctcttgcagtcgtcagcctgaacataacat
ccttgggattacgctccctcaaggagataagtgatggagatgtgataatttcaggaaa
caaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctccggt
cagaaaaccaaaattataagcaacagaggtgaaaacagctgcaaggccacaggccagg
tctgccatgccttgtgctcccccgagggctgctggggcccggagcccagggactgcgt
ctcttgccggaatgtcagccgaggcagggaatgcgtggacaagtgcaaccttctggag
ggtgagccaagggagtttgtggagaactctgagtgcatacagtgccacccagagtgcc
tgcctcaggccatgaacatcacctgcacaggacggggaccagacaactgtatccagtg
tgcccactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatggga
gaaaacaacaccctggtctggaagtacgcagacgccggccatgtgtgccacctgtgcc
atccaaactgcacctacggatgcactgggccaggtcttgaaggctgtccaacgaatgg
gcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtg
gtggccctggggatcggcctcttcatgtga 152
atgctgctcctcgtgacaagcctgctcctgtgtgaactccctcatccagcttttctgc
truncated
tcattcctcggaaagtgtgcaacggcatcggcatcggagagttcaaggacagcctgag EGFR
catcaatgccaccaacatcaagcacttcaagaattgcaccagcatcagcggcgacctg (tEGFR)
cacattctgcctgtggcctttagaggcgacagcttcacccacacacctccactggatc sequence
(nt) cccaagagctggatatcctgaaaaccgtgaaagagattaccggattcctcctgatcca
(O/SSE) agcctggccagagaacagaaccgatctgcacgccttcgagaacctcgagatcatcaga
ggccggaccaaacagcacggccagtttagcctggctgtggtgtctctgaacatcacca
gtctgggcctgagaagcctgaaagaaatctccgacggcgacgtgatcatctccggaaa
caagaacctgtgctacgccaacaccatcaactggaagaagctgttcggcacctccggc
cagaaaacaaagatcatctctaaccggggcgagaacagctgcaaggccaccggacaag
tttgtcacgccctgtgtagccctgaaggctgttggggacccgaacctagagactgtgt
gtcctgccggaatgtgtcccggggcagagaatgtgtggataagtgcaacctgctggaa
ggcgagccccgcgagtttgtggaaaacagcgagtgcatccagtgtcaccccgagtgtc
tgccccaggccatgaacattacatgcaccggcagaggccccgacaactgtattcagtg
cgcccactacatcgacggccctcactgcgtgaaaacatgtccagctggcgtgatggga
gagaacaacaccctcgtgtggaagtatgccgacgccggacatgtgtgccacctgtgtc
accctaattgcacctacggctgtaccggacctggcctggaaggatgccctacaaacgg
ccctaagatccccagcattgccaccggaatggttggagccctgctgcttctgttggtg
gtggccctcggaatcggcctgttcatgtga 153
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDL
truncated
HILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIR EGFR
GRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSG (tEGFR)
QKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE sequence
(aa) GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMG
ENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLV
VALGIGLFM 154
aagcgggggagaaagaaactgctgtatattttcaaacagccctttatgagacctgtgc 4-1BB
agactacccaggaggaagacggatgcagctgtaggtttcccgaggaagaggaaggagg
intracellular ctgtgagctg co-signaling sequence (nt) 155
aagcggggcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgc 4-1BB
agaccacacaagaggaagatggctgctcctgcagattccccgaggaagaagaaggcgg
intracellular ctgcgagctg co-signaling sequence (nt)- (O/SSE) 156
caggttcagctgcaagagtctggccctggcctggtcaagcctagcgaaacactgagcc
anti-ROR1
tgacctgtaccgtgtctggcggcagcatctccaactactactggtcctggatcagaca CAR (nt)
gcctgccggcaaaggcctggaatggatcggcagaatctacaccagcggcagcaccaac
tacaaccccagcctgaagtccagagtgaccatgagcgtggacaccagcaagaaccagt
tctccctgaagctgagcagcctgacagccgccgataccgccatctactactgtgcccg
gtactacgatatcctgaccggcttcttcgactactggggccagggaacactggtcaca
gtttctagcggaggcggaggatctggtggcggaggaagtggcggaggcggttctgtga
tttggatgacacagagccctagcctgctgagcgccagcacaggcgatagcgtgaccat
cagctgcagaatgagccaggacatcagcagctacctggcttggtatcagcagaagcct
ggcaaggcccctgaactgctgatctatgccgcttccagtctgcagagcggcgtgccat
ctagattttccggcagcggctctggcaccgacttcaccctgacaatcagctccctgca
gtccgaggacttcgccacctactattgccagcagtacgacagcttccctccaaccttt
ggccagggcaccaaggtggaattcaagcgcgagtctaaatacggaccgccttgtcctc
cttgtcccatgttctgggtgctcgtggtcgttggcggagtgctggcctgttacagcct
gctggttaccgtggccttcatcatcttttgggtcaagcggggcagaaagaagctgctc
tacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagaggaagatggct
gctcctgcagattccccgaggaagaagaaggcggctgcgagctgagagtgaagttcag
cagatccgccgacgctccagcctatcagcagggccaaaaccagctgtacaacgagctg
aacctggggagaagagaagagtacgacgtgctggataagcggagaggcagagatcctg
aaatgggcggcaagcccagacggaagaatcctcaagagggcctgtataatgagctgca
gaaagacaagatggccgaggcctacagcgagatcggaatgaagggcgagcgcagaaga
ggcaagggacacgatggactgtaccagggcctgagcaccgccaccaaggatacctatg
acgcactgcacatgcaggccctgccacctaga 157
cagctccagctgcaagaatctggacctggcctggtcaagcccagcgagacactgtctc
anti-ROR1
tgacctgtacagtgtccggcggcagcatcaatagcaccacaagctactgggcctggat CAR (nt)
cagacagcctcctggcaaaggcctggaatggatcggcaccatcttctacagcggcaag
acctacaacaaccccagcctgaagtccagagtgaccatgagcgtggacaccagcaaga
accacttcagcctgaaagtgaacagcgtgacagccgccgataccgccgtgtactactg
cgccagattcgactacggcttccacgacgccttcgacatctggggccagggcacaatg
gtcacagtttctagcggaggcggaggatctggtggcggaggaagtggcggaggcggtt
ctgagattgtgatgacacagagccccgccactctgagccttagtcctggcgaaagagc
cacactgagctgcagagccagccagagcatcaccagcgattacctgagctggtatcag
cagaagcccggacaggctcccagactgctgatctatggcgcctctacaagagccaccg
gcattcccgcccgcttttctggctctggaagcggcaccgacttcaccctgaccatatc
tagcctgcagcctgaggacttcgtggtgtactattgccagcaggactacaacctgtac
accttcggccaggggaccaagctggaaatcaagagagagtctaaatacggaccgcctt
gtcctccttgtcccatgttctgggtgctcgtggtcgttggcggagtgctggcctgtta
cagcctgctggttaccgtggccttcatcatcttttgggtcaagcggggcagaaagaag
ctgctctacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagaggaag
atggctgctcctgcagattccccgaggaagaagaaggcggctgcgagctgagagtgaa
gttcagcagatccgccgacgctccagcctatcagcagggccaaaaccagctgtacaac
gagctgaacctggggagaagagaagagtacgacgtgctggataagcggagaggcagag
atcctgaaatgggcggcaagcccagacggaagaatcctcaagagggcctgtataatga
gctgcagaaagacaagatggccgaggcctacagcgagatcggaatgaagggcgagcgc
agaagaggcaagggacacgatggactgtaccagggcctgagcaccgccaccaaggata
cctatgacgcactgcacatgcaggccctgccacctaga 158
caggttcagctgcaagagtctggccctggcctggtcaagcctagcgaaacactgagcc
anti-ROR1
tgacctgtaccgtgtctggcggcagcatctccaactactactggtcctggatcagaca CAR (nt)
gcctgccggcaaaggcctggaatggatcggcagaatctacaccagcggcagcaccaac
tacaaccccagcctgaagtccagagtgaccatgagcgtggacaccagcaagaaccagt
tctccctgaagctgagcagcctgacagccgccgataccgccatctactactgtgcccg
gtactacgatatcctgaccggcttcttcgactactggggccagggaacactggtcaca
gtttctagcggaggcggaggatctggtggcggaggaagtggcggaggcggttctgtga
tttggatgacacagagccctagcctgctgagcgccagcacaggcgatagcgtgaccat
cagctgcagaatgagccaggacatcagcagctacctggcttggtatcagcagaagcct
ggcaaggcccctgaactgctgatctatgccgcttccagtctgcagagcggcgtgccat
ctagattttccggcagcggctctggcaccgacttcaccctgacaatcagctccctgca
gtccgaggacttcgccacctactattgccagcagtacgacagcttccctccaaccttt
ggccagggcaccaaggtggaattcaagcgcgagtctaaatacggaccgccttgtcctc
cttgtcccggccagccaagagagccccaggtttacacactgcctccaagccaagagga
aatgaccaagaatcaggtgtccctgacatgcctggtcaagggcttctacccctccgat
atcgccgtggaatgggagagcaatggccagcctgagaacaactacaagaccacacctc
ctgtgctggacagcgacggcagtttcttcctgtatagtagactcaccgtggataaatc
aagatggcaagagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaac
cactacacccagaaaagcctgagcctgtctctgggcaagatgttctgggtgctcgtgg
tcgttggcggagtgctggcctgttacagcctgctggttaccgtggccttcatcatctt
ttgggtcaagcggggcagaaagaagctgctctacatcttcaagcagcccttcatgcgg
cccgtgcagaccacacaagaggaagatggctgctcctgcagattccccgaggaagaag
aaggcggctgcgagctgagagtgaagttcagcagatccgccgacgctccagcctatca
gcagggccaaaaccagctgtacaacgagctgaacctggggagaagagaagagtacgac
gtgctggataagcggagaggcagagatcctgaaatgggcggcaagcccagacggaaga
atcctcaagagggcctgtataatgagctgcagaaagacaagatggccgaggcctacag
cgagatcggaatgaagggcgagcgcagaagaggcaagggacacgatggactgtaccag
ggcctgagcaccgccaccaaggatacctatgacgcactgcacatgcaggccctgccac ctaga
159 caggtgcagctggttcaatctggcgccgaagtgaagaaaccaggcgcctctgtgaagg
anti-ROR1
tgtcctgcaaggccagcggctacacctttaccagctacggcatcagctgggtccgaca CAR (nt)
ggctcctggacaaggcttggaatggatgggctggatcagcgcctacaacggcaacacc
aaatacgcccagaaactgcagggcagagtgaccatgaccaccgacaccagcacaagca
ccgcctacatggaactgcggagcctgagatccgatgacaccgccgtgtactactgcgc
cagagatgaggacatcctgaccggctacaactactacggcatggacgtgtggggccag
ggcacaacagtgacagtttcttctggcggcggaggatctggcggaggtggaagcggag
gcggtggatctcaactggtgctgacacagtctcctagcgcctctgcttctctgggagc
cagcgtgaagctgacctgtacactgtctagcggccacagcagctacgccattgcttgg
catcagcagcagcccgagaagggccctagatacctgatgaagctgaacagcgacggca
gccactctaaaggcgacggcatccccgatagattcagcggcagttctagcggagccga
gcgctacctgacaatcagctctctgcaatccgaggacgaggccgactactactgtcag
acatggggcaccggcatcagagtgtttggcggaggcaccaagctgacagtgcttggag
agtctaaatacggaccgccttgtcctccttgtcccggccagccaagagagccccaggt
ttacacactgcctccaagccaagaggaaatgaccaagaatcaggtgtccctgacatgc
ctggtcaagggcttctacccctccgatatcgccgtggaatgggagagcaatggccagc
ctgagaacaactacaagaccacacctcctgtgctggacagcgacggcagtttcttcct
gtatagtagactcaccgtggataaatcaagatggcaagagggcaacgtgttcagctgc
agcgtgatgcacgaggccctgcacaaccactacacccagaaaagcctgagcctgtctc
tgggcaagatgttctgggtgctcgtggtcgttggcggagtgctggcctgttacagcct
gctggttaccgtggccttcatcatcttttgggtcaagcggggcagaaagaagctgctc
tacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagaggaagatggct
gctcctgcagattccccgaggaagaagaaggcggctgcgagctgagagtgaagttcag
cagatccgccgacgctccagcctatcagcagggccaaaaccagctgtacaacgagctg
aacctggggagaagagaagagtacgacgtgctggataagcggagaggcagagatcctg
aaatgggcggcaagcccagacggaagaatcctcaagagggcctgtataatgagctgca
gaaagacaagatggccgaggcctacagcgagatcggaatgaagggcgagcgcagaaga
ggcaagggacacgatggactgtaccagggcctgagcaccgccaccaaggatacctatg
acgcactgcacatgcaggccctgccacctaga 160
cagctccagctgcaagaatctggacctggcctggtcaagcccagcgagacactgtctc
anti-ROR1
tgacctgtacagtgtccggcggcagcatcaatagcaccacaagctactgggcctggat CAR (nt)
cagacagcctcctggcaaaggcctggaatggatcggcaccatcttctacagcggcaag
acctacaacaaccccagcctgaagtccagagtgaccatgagcgtggacaccagcaaga
accacttcagcctgaaagtgaacagcgtgacagccgccgataccgccgtgtactactg
cgccagattcgactacggcttccacgacgccttcgacatctggggccagggcacaatg
gtcacagtttctagcggaggcggaggatctggtggcggaggaagtggcggaggcggtt
ctgagattgtgatgacacagagccccgccactctgagccttagtcctggcgaaagagc
cacactgagctgcagagccagccagagcatcaccagcgattacctgagctggtatcag
cagaagcccggacaggctcccagactgctgatctatggcgcctctacaagagccaccg
gcattcccgcccgcttttctggctctggaagcggcaccgacttcaccctgaccatatc
tagcctgcagcctgaggacttcgtggtgtactattgccagcaggactacaacctgtac
accttcggccaggggaccaagctggaaatcaagagagagtctaaatacggaccgcctt
gtcctccttgtcccggccagccaagagagccccaggtttacacactgcctccaagcca
agaggaaatgaccaagaatcaggtgtccctgacatgcctggtcaagggcttctacccc
tccgatatcgccgtggaatgggagagcaatggccagcctgagaacaactacaagacca
cacctcctgtgctggacagcgacggcagtttcttcctgtatagtagactcaccgtgga
taaatcaagatggcaagagggcaacgtgttcagctgcagcgtgatgcacgaggccctg
cacaaccactacacccagaaaagcctgagcctgtctctgggcaagatgttctgggtgc
tcgtggtcgttggcggagtgctggcctgttacagcctgctggttaccgtggccttcat
catcttttgggtcaagcggggcagaaagaagctgctctacatcttcaagcagcccttc
atgcggcccgtgcagaccacacaagaggaagatggctgctcctgcagattccccgagg
aagaagaaggcggctgcgagctgagagtgaagttcagcagatccgccgacgctccagc
ctatcagcagggccaaaaccagctgtacaacgagctgaacctggggagaagagaagag
tacgacgtgctggataagcggagaggcagagatcctgaaatgggcggcaagcccagac
ggaagaatcctcaagagggcctgtataatgagctgcagaaagacaagatggccgaggc
ctacagcgagatcggaatgaagggcgagcgcagaagaggcaagggacacgatggactg
taccagggcctgagcaccgccaccaaggatacctatgacgcactgcacatgcaggccc
tgccacctaga 161
caggttcagctgcttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaagg
anti-ROR1
tgtcctgcaaggccagcggctacacctttaccagctacggcatcagctgggtccgaca CAR (nt)
ggctcctggacaaggcttggaatggatgggctggatcagcgcctacaccggcaatacc
agatacgcccagaaactgcagggcagagtgaccatgaccaccgacaccagcacaagca
ccgcctacatggaactgcggagcctgagatccgatgacaccgccgtgtactactgcgc
cagagaagaaggcgccaccaccgactacgactactacggcatggatgtgtggggccag
ggaacagccgtgacagtttcttctggtggcggaggatctggcggaggtggaagcggcg
gaggcggatctcaactggttctgacacagagcccaagcgcctctgcatctctgggagc
ttccgtgaagctgacctgcacactgtctagcggccacagcagctatgccattgcctgg
catcagcaacagcccgagaagggccctagatacctgatgaagctgaacagcgacggca
gccactctaaaggcgacggcatccccgatagattcagcggcagttctagcggagccga
gcgctacctgacaatcagctctctgcaatccgaggacgaggccgattactactgtcag
acatggggcaccggcatcagagtgtttggcggcggaacaaagctgaccgtgctgggcg
agtctaaatacggaccgccttgtcctccttgtcccggccagccaagagagccccaggt
ttacacactgcctccaagccaagaggaaatgaccaagaatcaggtgtccctgacatgc
ctggtcaagggcttctacccctccgatatcgccgtggaatgggagagcaatggccagc
ctgagaacaactacaagaccacacctcctgtgctggacagcgacggcagtttcttcct
gtatagtagactcaccgtggataaatcaagatggcaagagggcaacgtgttcagctgc
agcgtgatgcacgaggccctgcacaaccactacacccagaaaagcctgagcctgtctc
tgggcaagatgttctgggtgctcgtggtcgttggcggagtgctggcctgttacagcct
gctggttaccgtggccttcatcatcttttgggtcaagcggggcagaaagaagctgctc
tacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagaggaagatggct
gctcctgcagattccccgaggaagaagaaggcggctgcgagctgagagtgaagttcag
cagatccgccgacgctccagcctatcagcagggccaaaaccagctgtacaacgagctg
aacctggggagaagagaagagtacgacgtgctggataagcggagaggcagagatcctg
aaatgggcggcaagcccagacggaagaatcctcaagagggcctgtataatgagctgca
gaaagacaagatggccgaggcctacagcgagatcggaatgaagggcgagcgcagaaga
ggcaagggacacgatggactgtaccagggcctgagcaccgccaccaaggatacctatg
acgcactgcacatgcaggccctgccacctaga 162 SRGGGGSGGGGSGGGGSLEMA linker
163 GSRGGGGSGGGGSGGGGSLEMA linker 164
ggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacatcgoccacagtccc EF1alpha
cgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggg promoter
with gtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggag
HTLV1 aaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgc
enhancer cagaacacagctgaagcttcgaggggctcgcatctctccttcacgcgcccgccgccct
acctgaggccgccatccacgccggttgagtcgcgttctgccgcctcccgcctgtggtg
cctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccgggcct
ttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctg
accctgcttgctcaactctacgtctttgtttcgttttctgttctgcgccgttacagat
ccaagctgtgaccggcgcctac 165
aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttg
Woodchuck
ctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttc
Hepatitis
ccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgag Virus
(WHP) gagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaa
Posttranscriptional
cccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgcttt
Regulatory
ccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggaca
Element
ggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcct (WPRE)
ttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgcta
cgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctg
cggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccg
cctccccgc 166 GSGEGRGSLLTCGDVEENPGP T2A peptide (aa) 167
GSGQCTNYALLKLAGDVESNPGP E2A peptide (aa) 168
GSGVKQTLNFDLLKLAGDVESNPGP F2A peptide (aa) 169
ctcgagggcggcggagagggcagaggaagtcttctaacatgcggtgacgtggaggaga T2A
peptide atcccggccctagg (nt) 170
gagctggagcagccgccaccgccgccgccgagggagccccgggacggcagcccctggg human
ROR1 cgcagggtgcgctgttctcggagtccgacccagggcgactcacgcccactggtgcgac
(nt) ccggacagcctgggactgacccgccggcccaggcgaggctgcagccagagggctggga
GenBank: agggatcgcgctcgcggcatccagaggcggccaggcggaggcgagggagcaggttaga
M97675.1 gggacaaagagctttgcagacgtccccggcgtcctgcgagcgccagcggccgggacga
ggcggccgggagcccgggaagagcccgtggatgttctgcgcgcggcctgggagccgcc
gccgccgccgcctcagcgagaggaggaatgcaccggccgcgccgccgcgggacgcgcc
cgccgctcctggcgctgctggccgcgctgctgctggccgcacgcggggctgctgccca
agaaacagagctgtcagtcagtgctgaattagtgcctacctcatcatggaacatctca
agtgaactcaacaaagattcttacctgacccttgatgaaccaatgaataacatcacca
cgtctctgggccagacagcagaactgcactgcaaagtctctgggaatccacctcccac
catccgctggttcaaaaatgatgctcctgtggtccaggagccccggaggctctccttt
cggtccaccatctatggctctcggctgcggattagaaacctcgacaccacagacacag
gctacttccagtgcgtggcaacaaacggcaaggaggtggtttcttccactggagtctt
gtttgtcaagtttggcccccctcccactgcaagtccaggatactcagatgagtatgaa
gaagatggattctgtcagccatacagagggattgcatgtgcaagatttattggcaacc
gcaccgtctatatggagtctttgcacatgcaaggggaaatagaaaatcagatcacagc
tgccttcactatgattggcacttccagtcacttatctgataagtgttctcagttcgcc
attccttccctgtgccactatgccttcccgtactgcgatgaaacttcatccgtcccaa
agccccgtgacttgtgtcgcgatgaatgtgaaatcctggagaatgtcctgtgtcaaac
agagtacatttttgcaagatcaaatcccatgattctgatgaggctgaaactgccaaac
tgtgaagatctcccccagccagagagcccagaagctgcgaactgtatccggattggaa
ttcccatggcagatcctataaataaaaatcacaagtgttataacagcacaggtgtgga
ctaccgggggaccgtcagtgtgaccaaatcagggcgccagtgccagccatggaattcc
cagtatccccacacacacactttcaccgcccttcgtttcccagagctgaatggaggcc
attcctactgccgcaacccagggaatcaaaaggaagctccctggtgcttcaccttgga
tgaaaactttaagtctgatctgtgtgacatcccagcttgcgattcaaaggattccaag
gagaagaataaaatggaaatcctgtacatactagtgccaagtgtggccattcccctgg
ccattgctttactcttcttcttcatttgcgtctgtcggaataaccagaagtcatcgtc
ggcaccagtccagaggcaaccaaaacacgtcagaggtcaaaatgtggagatgtcaatg
ctgaatgcatataaacccaagagcaaggctaaagagctacctctttctgctgtacgct
ttatggaagaattgggtgagtgtgcctttggaaaaatctataaaggccatctctatct
cccaggcatggaccatgctcagctggttgctatcaagaccttgaaagactataacaac
ccccagcaatggatggaatttcaacaagaagcctccctaatggcagaactgcaccacc
ccaatattgtctgccttctaggtgccgtcactcaggaacaacctgtgtgcatgctttt
tgagtatattaatcagggggatctccatgagttcctcatcatgagatccccacactct
gatgttggctgcagcagtgatgaagatgggactgtgaaatccagcctggaccacggag
attttctgcacattgcaattcagattgcagctggcatggaatacctgtctagtcactt
ctttgtccacaaggaccttgcagctcgcaatattttaatcggagagcaacttcatgta
aagatttcagacttggggctttccagagaaatttactccgctgattactacagggtcc
agagtaagtccttgctgcccattcgctggatgccccctgaagccatcatgtatggcaa
attctcttctgattcagatatctggtcctttggggttgtcttgtgggagattttcagt
tttggactccagccatattatggattcagtaaccaggaagtgattgagatggtgagaa
aacggcagctcttaccatgctctgaagactgcccacccagaatgtacagcctcatgac
agagtgctggaatgagattccttctaggagaccaagatttaaagatattcacgtccgg
cttcggtcctgggagggactctcaagtcacacaagctctactactccttcagggggaa
atgccaccacacagacaacctccctcagtgccagcccagtgagtaatctcagtaaccc
cagatatcctaattacatgttcccgagccagggtattacaccacagggccagattgct
ggtttcattggcccgccaatacctcagaaccagcgattcattcccatcaatggatacc
caatacctcctggatatgcagcgtttccagctgcccactaccagccaacaggtcctcc
cagagtgattcagcactgcccacctcccaagagtcggtccccaagcagtgccagtggg
tcgactagcactggccatgtgactagcttgccctcatcaggatccaatcaggaagcaa
atattcctttactaccacacatgtcaattccaaatcatcctggtggaatgggtatcac
cgtttttggcaacaaatctcaaaaaccctacaaaattgactcaaagcaagcatcttta
ctaggagacgccaatattcatggacacaccgaatctatgatttctgcagaactgtaaa
atgcacaacttttgtaaatgtggtatacaggacaaactagacggccgtagaaaagatt
tatattcaaatgtttttattaaagtaaggttctcatttagcagacatcgcaacaagta
ccttctgtgaagtttcactgtgtcttaccaagcaggacagacactcggccag 171
QETELSVSAELVPTSSWNTSSEIDKGSYLTLDEPMNNITTSLGQTAELHCKVSGNPPP Mouse
SIRWFKNDAPVVQEPRRISFRATNYGSRLRIRNLDTTDTGYFQCVATNGKKVVSTTGV ROR1;
LFVKFGPPPTASPGSSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQIT GenBank
No. AAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEVLENVLCQ
NP_038873
TEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGV
DYRGTVSVTKSGRQCQPWNSQYPHTHSFTALRFPELNGGHSYCRNPGNQKEAPWCFTL
DENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLATAFLEFFICVCRNNQKSS
SPPVQRQPKPVRGQNVEMSMLNAYKPKSKAKELPLSAVREMEELGECTEGKIYKGHLY
LPGMDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCML
FEYMNQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSH
FFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSSLPIRWMPPEAIMYG
KFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSEDCPPRMYSLM
TECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLSN
PRFPNYMFPSQGITPQGQIAGFIGPAIPQNQRFIPINGYPIPPGYAAFPAAHYQPAGP
PRVIQHCPPPKSRSPSSASGSTSTGHVASLPSSGSNQEANVPLLPHMSIPNHPGGMGI
TVFGNKSQKPYKIDSKQSSLLGDSHIHGHTESMISAEV 172
QVQLQQSGAELVRPGASVTLSCKASGYTFSDYEMHWVIQTPVHGLEWIGAIDPETGGT 2A2
V.sub.H AYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTGYYDYDSFTYWGQGTLVTV
SA 173 DIVMTQSQKIMSTTVGDRVS1TCKASQNVDAAVAWYQQKPGQSPKLLIYSASNRYTGV
2A2 V.sub.L PDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYDIYPYTFGGGTKLEIK 174
QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWVRQAPGQGLEWMGSFDPYDGGS 99961
SYNQKFKDRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGHGTLVTVSS
humanized V.sub.H 175
QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWVRQAPGQGLEWMGSFDPYDGGS 99961
SYNQKFKDRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGHGTLVTVSS
humanized V.sub.H 176
QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWIRQPPGKGLEWIGSFDPYDGGS 99961
SYNQKFKDRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGHGTLVTVSS
humanized V.sub.H 177
QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWIRQPPGKGLEWIGSFDPYDGGS 99961
SYNQKFKDRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGHGTLVTVSS
humanized V.sub.H 178
DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPRLLIYSGSTLQSGT 99961
PPRFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIK humanized V.sub.L
179 DVVMTQSPLSLPVTLGQPASISCRASKSISKYLAWYQQKPGKAPKLLIYSGSTLQSGT
99961 PPRFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIK humanized
V.sub.L 180
DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPRLLIYSGSTLQSGT 99961
PPRFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIK humanized V.sub.L
181 DVVMTQSPLSLPVTLGQPASISCRASKSISKYLAWYQQKPGKAPKLLIYSGSTLQSGT
99961 PPRFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIK humanized
V.sub.L 182
agagtcaagttttccaggtccgccgacgctccagcctaccagcaggggcagaaccagc CD3-zeta
tgtacaacgagctgaacctgggcagaagggaagagtacgacgtcctggataagcggag derived
aggccgggaccctgagatgggcggcaagcctcggcggaagaacccccaggaaggcctg
intracellular
tataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagg
signaling
gcgagcggaggcggggcaagggccacgacggcctgtatcagggcctgtccaccgccac domain
(nt) caaggatacctacgacgccctgcacatgcaggccctgcccccaagg 183
aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgcc CD28
ccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagccta
endodomain tcgctcc (nt) 184
QVQLQESGPGLVKPSETLSLTCTVSGGSISNYYWSWIRQPAGKGLEWIGRIYTSGSTN
anti-ROR1
YNPSLKSRVTMSVDTSKNQFSLKLSSLTAADTAIYYCARYYDILTGFEDYWGQGTLVT CAR (aa)
VSSGGGGSGGGGSGGGGSVIWMTQSPSLLSASTGDSVTISCRMSQDISSYLAWYQQKP
GKAPELLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYYCQQYDSFPPTF
GQGTKVEFKRESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLL
YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL
NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR
GKGHDGLYQGLSTATKDTYDALHMQALPPR 185
QLQLQESGPGLVKPSETLSLTCTVSGGSINSTTSYWAWIRQPPGKGLEWIGTIFYSGK
anti-ROR1
TYNNPSLKSRVTMSVDTSKNHFSLKVNSVTAADTAVYYCARFDYGEHDAFDIWGQGTM CAR (aa)
VTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQSITSDYLSWYQ
QKPGQAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISSLQPEDFVVYYCQQDYNLY
TFGQGTKLEIKRESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKK
LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYN
ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER
RRGKGHDGLYQGLSTATKDTYDALHMQALPPR 186
QVQLQESGPGLVKPSETLSLTCTVSGGSISNYYWSWIRQPAGKGLEWIGRIYTSGSTN
anti-ROR1
YNPSLKSRVTMSVDTSKNQFSLKLSSLTAADTAIYYCARYYDILTGFEDYWGQGTLVT CAR (aa)
VSSGGGGSGGGGSGGGGSVIWMTQSPSLLSASTGDSVTISCRMSQDISSYLAWYQQKP
GKAPELLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYYCQQYDSFPPTF
GQGTKVEFKRESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFELYSRLTVDKSRWQEGNVFSCSVMHEALHN
HYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMR
PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD
VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
GLSTATKDTYDALHMQALPPR 187
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNT
anti-ROR1
KYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDEDILTGYNYYGMDVWGQ CAR (aa)
GTTVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSGHSSYAIAW
HQQQPEKGPRYLMKLNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCQ
TWGTGIRVFGGGTKLTVLGESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSRLTVDKSRWQEGNVFSC
SVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLL
YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL
NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR
GKGHDGLYQGLSTATKDTYDALHMQALPPR 188
QLQLQESGPGLVKPSETLSLTCTVSGGSINSTTSYWAWIRQPPGKGLEWIGTIFYSGK
anti-ROR1
TYNNPSLKSRVTMSVDTSKNHFSLKVNSVTAADTAVYYCARFDYGEHDAFDIWGQGTM CAR (aa)
VTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQSITSDYLSWYQ
QKPGQAPRLLIYGASTRATGIPARESGSGSGTDFTLTISSLQPEDFVVYYCQQDYNLY
TFGQGTKLEIKRESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSRLTVDKSRWQEGNVFSCSVMHEAL
HNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPF
MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREE
YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL
YQGLSTATKDTYDALHMQALPPR 189
QVQLLQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYTGNT
anti-ROR1
RYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAREEGATTDYDYYGMDVWGQ CAR (aa)
GTAVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSGHSSYAIAW
HQQQPEKGPRYLMKLNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCQ
TWGTGIRVFGGGTKLTVLGESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSRLTVDKSRWQEGNVFSC
SVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLL
YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL
NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR
GKGHDGLYQGLSTATKDTYDALHMQALPPR 190
atgcctctgctgctgcttctgcctcttctttgggctggtgctctggct CD33 signal
sequence (nt) (O/SSE) 191
atgccgctgctgctactgctgcccctgctgtgggcaggggccctggct CD33 signal
sequence (nt) GenBank: M23197.1 192
gaatctaagtacggaccgccctgccccccttgccctatg Spacer (IgG4 hinge)(nt) 193
GAATCTAAGTACGGACCGCCTTGTCCTCCATGTCCTGGCCAGCCAAGAGAACCCCAGG
Hinge-C.sub.H3
TGTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTG spacer
(nt) CCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAG
CCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCC
TGTACAGCCGGCTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTG
CAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGAGC
CTGGGCAAG 194
ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW
IgG4/IgG2
YVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT hinge-
ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
IgG2/IgG4 TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM
C.sub.H2-IgG4 C.sub.H3 spacer (aa) 195
gaatctaagtacggaccgccctgccctccctgccctgctcctcctgtggctggaccaa
IgG4/IgG2
gcgtgttcctgtttccacctaagcctaaagataccctgatgatttcccgcacacctga hinge-
agtgacttgcgtggtcgtggacgtgagccaggaggatccagaagtgcagttcaactgg
IgG2/IgG4
tacgtggacggcgtggaagtccacaatgctaagactaaaccccgagaggaacagtttc
C.sub.H2-IgG4
agtcaacttaccgggtcgtgagcgtgctgaccgtcctgcatcaggattggctgaacgg C.sub.H3
spacer gaaggagtataagtgcaaagtgtctaataagggactgcctagctccatcgagaaaaca
(nt) attagtaaggcaaaagggcagcctcgagaaccacaggtgtataccctgccccctagcc
aggaggaaatgaccaagaaccaggtgtccctgacatgtctggtcaaaggcttctatcc
aagtgacatcgccgtggagtgggaatcaaatgggcagcccgagaacaattacaagacc
acaccacccgtgctggactctgatggaagtttctttctgtattccaggctgaccgtgg
ataaatctcgctggcaggagggcaacgtgttctcttgcagtgtcatgcacgaagccct
gcacaatcattatacacagaagtcactgagcctgtccctgggcaaaatg
196 gagtctaaatacggaccgccttgtcctccttgtcccgctcctcctgttgccggacctt
IgG4/IgG2
ccgtgttcctgtttcctccaaagcctaaggacaccctgatgatcagcaggacccctga hinge-
agtgacctgcgtggtggtggatgtgtcccaagaggatcccgaggtgcagttcaactgg
IgG2/IgG4
tatgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagttcc
C.sub.H2-IgG4
agagcacctacagagtggtgtccgtgctgacagtgctgcaccaggattggctgaacgg C.sub.H3
spacer caaagagtacaagtgcaaggtgtccaacaagggcctgcctagcagcatcgagaaaacc
(nt) O/SSE
atctccaaggccaagggccagccaagagagccccaggtttacacactgcctccaagcc
aagaggaaatgaccaagaatcaggtgtccctgacatgcctggtcaagggcttctaccc
ctccgatatcgccgtggaatgggagagcaatggccagcctgagaacaactacaagacc
acacctcctgtgctggacagcgacggcagtttcttcctgtatagtagactcaccgtgg
ataaatcaagatggcaagagggcaacgtgttcagctgcagcgtgatgcacgaggccct
gcacaaccactacacccagaaaagcctgagcctgtctctgggcaagatg 197
ttttgggtgctggtcgtggtcggaggggtgctggcctgttacagcctgctggtgacag CD28
tcgctttcatcatcttctgggtg transmembrane domain (nt) 198
ttctgggtgctcgtggtcgttggcggagtgctggcctgttacagcctgctggttaccg CD28
tggccttcatcatcttttgggtc transmembrane domain (nt) (O/SSE) 199
FRSTIYGSRLRIRNL ROR1 epitope 1 200 LSVSAELVPTSSW ROR1 epitope 2 201
HCKVSGNPPPTIRW ROR1 epitope 3 202 WFKNDAPVVQEPRRLSFRSTIYGSRL ROR1
epitope 4 203 VSSTGVLFV ROR1 epitope 5 204 LFVKFGPPPTASP ROR1
epitope 6 205 DEYEEDGFCQP ROR1 epitope 7 206 QPYRGIACARFIGN ROR1
epitope 8 207 SQFAIPSLCHYAFP ROR1 epitope 9 208 AFPYCDETSSVP ROR1
epitope 10 209 NVLCQTEYIFARSNPMILMR ROR1 epitope 11 210
LKLPNCEDLPQP ROR1 epitope 12 211 PESPEAANCIRIGIP ROR1 epitope 13
212 VDYRGTVSVTKSGR ROR1 epitope 14 213 SQYPHTHTFTALRF ROR1 epitope
15 214 DSKEKNKMEILYILV ROR1 epitope 16 215
MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNISSELNKDSYLTLDEPM
human
NNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGY
(Homo
FQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESL
sapiens)
HMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILE
RORI
NVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVD
(Uniprot
YRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSD
Q01973)
LCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSAPVQRQPKHVRG
QNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHAQLVAIKTLKDYN
NPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYINQGDLHEFLIMRSPHSDVGCS
SDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREI
YSADYYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEM
VRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATT
QTTSLSASPVSNLSNPRYPNYMFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFP
AAHYQPTGPPRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGG
MGITVFGNKSQKPYKIDSKQASLLGDANIHGHTESMISAEL 216
MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNISSELNKDSYLTLDEPM
Rhesus
NNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGY
macaque
FQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESL
(Macaca
HMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILE
mulatta)
NVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVD
ROR1
YRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSD
(Uniprot
LCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSPPVQRQPKHVRG
F6RUP2)
QNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHAQLVAIKTLKDYN
NPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYMNQGDLHEFLIMRSPHSDVGCS
SDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREI
YSADYYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEM
VRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATT
QTTSLSASPVSNLSNPRYPNYIFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFP
AAHYQPTGPPRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGG
MGITVFGNKSQKPYKIDAKQASLLGDANIHGHTESMISAEL 217
MLRTAHKLLYLILPLSFSLPFFFFSETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITT
cynomolgus
SLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVA
macaque
TNGKEVVSSTGVLFVKFGKDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAF
(macaca
TMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSN
fasicularis)
PMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQC
ROR1
QPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKE
(Uniprot
KNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSPPVQRQPKHVRGQNVEMSMLNAYKPK
A0A2K5WT
SKAKELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHAQLVAIKTLKDYNNPQQWTEFQQEASL
X7)
MAELHHPNIVCLLGAVTQEQPVCMLFEYMNQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHG
DFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSLL
PIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSEDCP
PRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLS
NPRYPNYIFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFPAAHYQPTGPPRVIQ
HCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGGMGITVFGNKSQKPY
KIDAKQASLLGDANIHGHTESMISAEL 218
SYLTLDEPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIR
cynomolgus
NLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIG
macaque
NRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDL
(macaca
CRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNH
fasicularis)
KCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCF
ROR1
TLDENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSPPV
(Uniprot
QRQPKHVRGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHAQLV
A0A2K5WT
AIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYMNQGDLHEFLIMR
X4)
SPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKI
SDLGLSREIYSADYYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYG
FSNQEVIEMVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSST
TPSGGNATTQTTSLSASPVSNLSNPRYPNYIFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYP
IPPGYAAFPAAHYQPTGPPRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPH
MSIPNHPGGMGITVFGNKSQKPYKIDAKQASLLGDANIHGHTESMISAEL 219
MHRPRRRGTRPPPLALLAALLLAARGADAQETELSVSAELVPTSSWNTSSEIDKGSYLTLDEPM
mouse (mus
NNITTSLGQTAELHCKVSGNPPPSIRWFKNDAPVVQEPRRISFRATNYGSRLRIRNLDTTDTGY
musculus)
FQCVATNGKKVVSTTGVLFVKFGPPPTASPGSSDEYEEDGFCQPYRGIACARFIGNRTVYMESL
ROR1
HMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEVLE
(Uniprot
NVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVD
Q9Z139)
YRGTVSVTKSGRQCQPWNSQYPHTHSFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSD
LCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIAFLFFFICVCRNNQKSSSPPVQRQPKPVRG
QNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECTFGKIYKGHLYLPGMDHAQLVAIKTLKDYN
NPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYMNQGDLHEFLIMRSPHSDVGCS
SDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREI
YSADYYRVQSKSSLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEM
VRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATT
QTTSLSASPVSNLSNPRFPNYMFPSQGITPQGQIAGFIGPAIPQNQRFIPINGYPIPPGYAAFP
AAHYQPAGPPRVIQHCPPPKSRSPSSASGSTSTGHVASLPSSGSNQEANVPLLPHMSIPNHPGG
MGITVFGNKSQKPYKIDSKQSSLLGDSHIHGHTESMISAEV
Sequence CWU 1
1
219112PRTartificial sequenceSpacer (IgG4 hinge) 1Glu Ser Lys Tyr
Gly Pro Pro Cys Pro Pro Cys Pro1 5 10236DNAartificial
sequenceSpacer (IgG4 hinge) 2gaatctaagt acggaccgcc ctgcccccct
tgccct 363119PRTartificial sequenceHinge-CH3 spacer 3Glu Ser Lys
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg1 5 10 15Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 20 25 30Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 35 40
45Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
50 55 60Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser65 70 75 80Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser 85 90 95Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser 100 105 110Leu Ser Leu Ser Leu Gly Lys
1154357DNAartificial sequenceHinge-CH3 spacer 4gaatctaagt
acggaccgcc ttgtcctcca tgtcctggcc agccaagaga accccaggtg 60tacacactgc
ctccaagcca agaggaaatg accaagaacc aggtgtccct gacctgcctg
120gtcaagggct tctacccttc cgatatcgcc gtggaatggg agagcaatgg
ccagcctgag 180aacaactaca agaccacacc tcctgtgctg gacagcgacg
gctcattctt cctgtacagc 240cggctgaccg tggacaagag cagatggcaa
gagggcaacg tgttcagctg cagcgtgatg 300cacgaggccc tgcacaacca
ctacacccag aagtctctga gcctgagcct gggcaag 3575282PRTartificial
sequenceIgD-hinge-Fc 5Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser
Ser Val Pro Thr Ala1 5 10 15Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys
Ala Thr Thr Ala Pro Ala 20 25 30Thr Thr Arg Asn Thr Gly Arg Gly Gly
Glu Glu Lys Lys Lys Glu Lys 35 40 45Glu Lys Glu Glu Gln Glu Glu Arg
Glu Thr Lys Thr Pro Glu Cys Pro 50 55 60Ser His Thr Gln Pro Leu Gly
Val Tyr Leu Leu Thr Pro Ala Val Gln65 70 75 80Asp Leu Trp Leu Arg
Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly 85 90 95Ser Asp Leu Lys
Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val 100 105 110Pro Thr
Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly 115 120
125Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn
130 135 140Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu
Pro Pro145 150 155 160Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala
Gln Ala Pro Val Lys 165 170 175Leu Ser Leu Asn Leu Leu Ala Ser Ser
Asp Pro Pro Glu Ala Ala Ser 180 185 190Trp Leu Leu Cys Glu Val Ser
Gly Phe Ser Pro Pro Asn Ile Leu Leu 195 200 205Met Trp Leu Glu Asp
Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro 210 215 220Ala Arg Pro
Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser225 230 235
240Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr
245 250 255Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala
Ser Arg 260 265 270Ser Leu Glu Val Ser Tyr Val Thr Asp His 275
280624PRTartificial sequenceT2A 6Leu Glu Gly Gly Gly Glu Gly Arg
Gly Ser Leu Leu Thr Cys Gly Asp1 5 10 15Val Glu Glu Asn Pro Gly Pro
Arg 207357PRTartificial sequencetEGFR 7Met Leu Leu Leu Val Thr Ser
Leu Leu Leu Cys Glu Leu Pro His Pro1 5 10 15Ala Phe Leu Leu Ile Pro
Arg Lys Val Cys Asn Gly Ile Gly Ile Gly 20 25 30Glu Phe Lys Asp Ser
Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe 35 40 45Lys Asn Cys Thr
Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala 50 55 60Phe Arg Gly
Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu65 70 75 80Leu
Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile 85 90
95Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu
100 105 110Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser
Leu Ala 115 120 125Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg
Ser Leu Lys Glu 130 135 140Ile Ser Asp Gly Asp Val Ile Ile Ser Gly
Asn Lys Asn Leu Cys Tyr145 150 155 160Ala Asn Thr Ile Asn Trp Lys
Lys Leu Phe Gly Thr Ser Gly Gln Lys 165 170 175Thr Lys Ile Ile Ser
Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly 180 185 190Gln Val Cys
His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu 195 200 205Pro
Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys 210 215
220Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val
Glu225 230 235 240Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu
Pro Gln Ala Met 245 250 255Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp
Asn Cys Ile Gln Cys Ala 260 265 270His Tyr Ile Asp Gly Pro His Cys
Val Lys Thr Cys Pro Ala Gly Val 275 280 285Met Gly Glu Asn Asn Thr
Leu Val Trp Lys Tyr Ala Asp Ala Gly His 290 295 300Val Cys His Leu
Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro305 310 315 320Gly
Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala 325 330
335Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly
340 345 350Ile Gly Leu Phe Met 355827PRThomo
sapiensMISC_FEATURECD28 8Phe Trp Val Leu Val Val Val Gly Gly Val
Leu Ala Cys Tyr Ser Leu1 5 10 15Leu Val Thr Val Ala Phe Ile Ile Phe
Trp Val 20 25966PRThomo sapiensMISC_FEATURECD28 9Ile Glu Val Met
Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn1 5 10 15Gly Thr Ile
Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30Phe Pro
Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly 35 40 45Val
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 50 55
60Trp Val651041PRThomo sapiensMISC_FEATURECD28 10Arg Ser Lys Arg
Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr1 5 10 15Pro Arg Arg
Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30Pro Arg
Asp Phe Ala Ala Tyr Arg Ser 35 401141PRThomo
sapiensMISC_FEATURECD28 (LL to GG) 11Arg Ser Lys Arg Ser Arg Gly
Gly His Ser Asp Tyr Met Asn Met Thr1 5 10 15Pro Arg Arg Pro Gly Pro
Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30Pro Arg Asp Phe Ala
Ala Tyr Arg Ser 35 401242PRThomo sapiensMISC_FEATURE4-1BB 12Lys Arg
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met1 5 10 15Arg
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25
30Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 4013112PRThomo
sapiensMISC_FEATURECD3 zeta 13Arg Val Lys Phe Ser Arg Ser Ala Asp
Ala Pro Ala Tyr Gln Gln Gly1 5 10 15Gln Asn Gln Leu Tyr Asn Glu Leu
Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30Asp Val Leu Asp Lys Arg Arg
Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45Pro Arg Arg Lys Asn Pro
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60Asp Lys Met Ala Glu
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg65 70 75 80Arg Arg Gly
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95Thr Lys
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105
11014112PRThomo sapiensMISC_FEATURECD3 zeta 14Arg Val Lys Phe Ser
Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly1 5 10 15Gln Asn Gln Leu
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30Asp Val Leu
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45Pro Arg
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60Asp
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg65 70 75
80Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
Arg 100 105 11015112PRThomo sapiensMISC_FEATURECD3 zeta 15Arg Val
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly1 5 10 15Gln
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25
30Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
Lys 50 55 60Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
Glu Arg65 70 75 80Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
Leu Ser Thr Ala 85 90 95Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
Ala Leu Pro Pro Arg 100 105 11016335PRTartificial sequencetEGFR
16Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu1
5 10 15Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser
Ile 20 25 30Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp
Ser Phe 35 40 45Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile
Leu Lys Thr 50 55 60Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala
Trp Pro Glu Asn65 70 75 80Arg Thr Asp Leu His Ala Phe Glu Asn Leu
Glu Ile Ile Arg Gly Arg 85 90 95Thr Lys Gln His Gly Gln Phe Ser Leu
Ala Val Val Ser Leu Asn Ile 100 105 110Thr Ser Leu Gly Leu Arg Ser
Leu Lys Glu Ile Ser Asp Gly Asp Val 115 120 125Ile Ile Ser Gly Asn
Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp 130 135 140Lys Lys Leu
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn145 150 155
160Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu
165 170 175Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys
Val Ser 180 185 190Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp
Lys Cys Asn Leu 195 200 205Leu Glu Gly Glu Pro Arg Glu Phe Val Glu
Asn Ser Glu Cys Ile Gln 210 215 220Cys His Pro Glu Cys Leu Pro Gln
Ala Met Asn Ile Thr Cys Thr Gly225 230 235 240Arg Gly Pro Asp Asn
Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro 245 250 255His Cys Val
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr 260 265 270Leu
Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His 275 280
285Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro
290 295 300Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val
Gly Ala305 310 315 320Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile
Gly Leu Phe Met 325 330 3351718PRTartificial sequenceT2A 17Glu Gly
Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro1 5 10 15Gly
Pro1822PRTartificial sequenceP2A 18Gly Ser Gly Ala Thr Asn Phe Ser
Leu Leu Lys Gln Ala Gly Asp Val1 5 10 15Glu Glu Asn Pro Gly Pro
201919PRTartificial sequenceP2A 19Ala Thr Asn Phe Ser Leu Leu Lys
Gln Ala Gly Asp Val Glu Glu Asn1 5 10 15Pro Gly
Pro2020PRTartificial sequenceE2A 20Gln Cys Thr Asn Tyr Ala Leu Leu
Lys Leu Ala Gly Asp Val Glu Ser1 5 10 15Asn Pro Gly Pro
202122PRTartificial sequenceF2A 21Val Lys Gln Thr Leu Asn Phe Asp
Leu Leu Lys Leu Ala Gly Asp Val1 5 10 15Glu Ser Asn Pro Gly Pro
202210PRTartificial sequencelinkerREPEAT(5)..(9)SGGGG is repeated 5
times 22Pro Gly Gly Gly Ser Gly Gly Gly Gly Pro1 5
102317PRTartificial sequencelinker 23Gly Ser Ala Asp Asp Ala Lys
Lys Asp Ala Ala Lys Lys Asp Gly Lys1 5 10 15Ser2418PRTartificial
sequencelinker 24Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly
Glu Gly Ser Thr1 5 10 15Lys Gly255PRTartificial
sequencehingeVARIANT(1)..(1)X is glycine, cysteine or
arginineVARIANT(4)..(4)X is cysteine or threonine 25Xaa Pro Pro Xaa
Pro1 52615PRTartificial sequencehinge 26Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro1 5 10 152712PRTartificial
sequencehinge 27Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro1 5
102861PRTartificial sequencehinge 28Glu Leu Lys Thr Pro Leu Gly Asp
Thr His Thr Cys Pro Arg Cys Pro1 5 10 15Glu Pro Lys Ser Cys Asp Thr
Pro Pro Pro Cys Pro Arg Cys Pro Glu 20 25 30Pro Lys Ser Cys Asp Thr
Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro 35 40 45Lys Ser Cys Asp Thr
Pro Pro Pro Cys Pro Arg Cys Pro 50 55 602912PRTartificial
sequencehinge 29Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro1 5
103036DNAartificial sequenceSpacer (IgG4hinge) O/SSE 30gagtctaaat
acggaccgcc ttgtcctcct tgtccc 36319PRTartificial sequencehinge 31Tyr
Gly Pro Pro Cys Pro Pro Cys Pro1 53210PRTartificial sequencehinge
32Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro1 5 103314PRTartificial
sequencehinge 33Glu Val Val Val Lys Tyr Gly Pro Pro Cys Pro Pro Cys
Pro1 5 1034228PRTartificial sequenceHinge-CH2-CH3 spacer 34Glu Ser
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val1 5 10 15Ala
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25
30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
35 40 45Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu 50 55 60Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln
Ser Thr65 70 75 80Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn 85 90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu Pro Ser Ser 100 105 110Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln 115 120 125Val Tyr Thr Leu Pro Pro Ser
Gln Glu Glu Met Thr Lys Asn Gln Val 130 135 140Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155 160Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170
175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
180 185 190Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser Val 195 200 205Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 210
215 220Ser Leu Gly Lys22535684DNAartificial sequenceHinge-CH2-CH3
spacer 35gaatctaagt acggaccgcc ttgtcctcca tgtcctgctc ctccagttgc
cggaccttcc 60gtgttcctgt ttcctccaaa gcctaaggac accctgatga tcagcagaac
ccctgaagtg 120acctgcgtgg tggtggacgt gtcccaagag gatcctgagg
tgcagttcaa ctggtatgtg 180gacggcgtgg aagtgcacaa cgccaagacc
aagcctagag aggaacagtt ccagagcacc 240tacagagtgg tgtccgtgct
gacagtgctg caccaggatt ggctgaacgg caaagagtac 300aagtgcaagg
tgtccaacaa gggcctgcct agcagcatcg agaaaaccat cagcaaggcc
360aagggccagc caagagaacc ccaggtgtac acactgcctc caagccaaga
ggaaatgacc 420aagaaccagg tgtccctgac ctgcctggtc aagggcttct
acccttccga tatcgccgtg 480gaatgggaga gcaatggcca gcctgagaac
aactacaaga ccacacctcc tgtgctggac 540agcgacggct cattcttcct
gtacagccgg ctgaccgtgg acaagagcag atggcaagag 600ggcaacgtgt
tcagctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag
660tctctgagcc tgagcctggg caag 68436229PRTartificial
sequenceHinge-CH2-CH3 spacer 36Glu Ser Lys Tyr Gly Pro Pro Cys Pro
Pro Cys Pro Ala Pro Glu Phe1 5 10 15Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 35 40 45Ser Gln Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105
110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln 130 135 140Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala145 150 155 160Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190Thr Val Asp Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220Leu
Ser Leu Gly Lys22537228PRTartificial sequenceIgG4/IgG2 hinge-
IgG2/IgG4 CH2- IgG4 CH3 spacer 37Glu Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro Pro Val1 5 10 15Ala Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45Gln Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr65 70 75 80Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 100 105
110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
115 120 125Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
Gln Val 130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro 165 170 175Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr 180 185 190Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 195 200 205Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220Ser
Leu Gly Lys22538684DNAartificial sequenceIgG4/IgG2 hinge- IgG2/IgG4
CH2- IgG4 CH3 spacer 38gaatctaagt acggaccgcc ctgccctccc tgccctgctc
ctcctgtggc tggaccaagc 60gtgttcctgt ttccacctaa gcctaaagat accctgatga
tttcccgcac acctgaagtg 120acttgcgtgg tcgtggacgt gagccaggag
gatccagaag tgcagttcaa ctggtacgtg 180gacggcgtgg aagtccacaa
tgctaagact aaaccccgag aggaacagtt tcagtcaact 240taccgggtcg
tgagcgtgct gaccgtcctg catcaggatt ggctgaacgg gaaggagtat
300aagtgcaaag tgtctaataa gggactgcct agctccatcg agaaaacaat
tagtaaggca 360aaagggcagc ctcgagaacc acaggtgtat accctgcccc
ctagccagga ggaaatgacc 420aagaaccagg tgtccctgac atgtctggtc
aaaggcttct atccaagtga catcgccgtg 480gagtgggaat caaatgggca
gcccgagaac aattacaaga ccacaccacc cgtgctggac 540tctgatggaa
gtttctttct gtattccagg ctgaccgtgg ataaatctcg ctggcaggag
600ggcaacgtgt tctcttgcag tgtcatgcac gaagccctgc acaatcatta
tacacagaag 660tcactgagcc tgtccctggg caaa 684395PRTartificial
sequence4GS linker 39Gly Gly Gly Gly Ser1 5404PRTartificial
sequence3GS linker 40Gly Gly Gly Ser14115PRTartificial
sequence(4GS)3 linker 41Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser1 5 10 154216PRTartificial sequenceCD33 Signal
Peptide 42Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala
Leu Ala1 5 10 154320PRTartificial sequencehuman IgG-kappa signal
peptide 43Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp
Ile Ser1 5 10 15Gly Ala Tyr Gly 204460DNAartificial sequencehuman
IgG-kappa signal sequence 44atggtgctgc agacccaggt gttcatcagc
ctgctgctgt ggatctccgg agcatacgga 604522PRTartificial sequenceGMCSFR
alpha chain signal peptide 45Met Leu Leu Leu Val Thr Ser Leu Leu
Leu Cys Glu Leu Pro His Pro1 5 10 15Ala Phe Leu Leu Ile Pro
204666DNAartificial sequenceGMCSFR alpha chain signal sequence
46atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg
60atccca 664718PRTartificial sequenceCD8 alpha signal peptide 47Met
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10
15His Ala48326PRThomo sapiensMISC_FEATUREIgG2 Fc (Uniprot P01859)
48Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1
5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95Thr Val Glu Arg Lys Cys Cys Val Glu
Cys Pro Pro Cys Pro Ala Pro 100 105 110Pro Val Ala Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140Val Ser His
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly145 150 155
160Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
165 170 175Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln
Asp Trp 180 185 190Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro 195 200 205Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys Gly Gln Pro Arg Glu 210 215 220Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn225 230 235 240Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255Ser Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280
285Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
290 295 300Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu305 310 315 320Ser Leu Ser Pro Gly Lys 32549327PRThomo
sapiensMISC_FEATUREIgG4 Fc (Uniprot P01861) 49Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75
80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala
Pro 100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200
205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
Thr Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315
320Leu Ser Leu Ser Leu Gly Lys 325507PRTartificial sequenceCDR-H1
50Gly Tyr Thr Phe Thr Ser Tyr1 55110PRTartificial sequenceCDR-H1
51Gly Tyr Thr Phe Thr Ser Tyr Gly Ile Ser1 5 10525PRTartificial
sequenceCDR-H1 52Ser Tyr Gly Ile Ser1 5538PRTartificial
sequenceCDR-H1 53Gly Tyr Thr Phe Thr Ser Tyr Gly1 5546PRTARTIFICIAL
SEQUENCECDR-H2 54Ser Ala Tyr Asn Gly Asn1 55510PRTARTIFICIAL
SEQUENCECDR-H2 55Trp Ile Ser Ala Tyr Asn Gly Asn Thr Lys1 5
105617PRTARTIFICIAL SEQUENCECDR-H2 56Trp Ile Ser Ala Tyr Asn Gly
Asn Thr Lys Tyr Ala Gln Lys Leu Gln1 5 10 15Gly578PRTARTIFICIAL
SEQUENCECDR-H2 57Ile Ser Ala Tyr Asn Gly Asn Thr1
55815PRTARTIFICIAL SEQUENCECDR-H3 58Asp Glu Asp Ile Leu Thr Gly Tyr
Asn Tyr Tyr Gly Met Asp Val1 5 10 155917PRTARTIFICIAL
SEQUENCECDR-H3 59Ala Arg Asp Glu Asp Ile Leu Thr Gly Tyr Asn Tyr
Tyr Gly Met Asp1 5 10 15Val6012PRTARTIFICIAL SEQUENCECDR-L1 60Thr
Leu Ser Ser Gly His Ser Ser Tyr Ala Ile Ala1 5 10617PRTARTIFICIAL
SEQUENCECDR-L1 61Ser Gly His Ser Ser Tyr Ala1 56211PRTARTIFICIAL
SEQUENCECDR-L2 62Leu Asn Ser Asp Gly Ser His Ser Lys Gly Asp1 5
10637PRTARTIFICIAL SEQUENCECDR-L2 63Leu Asn Ser Asp Gly Ser His1
5649PRTARTIFICIAL SEQUENCECDR-L3 64Gln Thr Trp Gly Thr Gly Ile Arg
Val1 5657PRTARTIFICIAL SEQUENCECDR-H1 65Gly Gly Ser Ile Ser Asn
Tyr1 56610PRTARTIFICIAL SEQUENCECDR-H1 66Gly Gly Ser Ile Ser Asn
Tyr Tyr Trp Ser1 5 10675PRTARTIFICIAL SEQUENCECDR-H1 67Asn Tyr Tyr
Trp Ser1 5688PRTARTIFICIAL SEQUENCECDR-H1 68Gly Gly Ser Ile Ser Asn
Tyr Tyr1 5695PRTARTIFICIAL SEQUENCECDR-H2 69Tyr Thr Ser Gly Ser1
5709PRTARTIFICIAL SEQUENCECDR-H2 70Arg Ile Tyr Thr Ser Gly Ser Thr
Asn1 57116PRTARTIFICIAL SEQUENCECDR-H2 71Arg Ile Tyr Thr Ser Gly
Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser1 5 10 15727PRTARTIFICIAL
SEQUENCECDR-H2 72Ile Tyr Thr Ser Gly Ser Thr1 57311PRTARTIFICIAL
SEQUENCECDR-H3 73Tyr Tyr Asp Ile Leu Thr Gly Phe Phe Asp Tyr1 5
107413PRTARTIFICIAL SEQUENCECDR-H3 74Ala Arg Tyr Tyr Asp Ile Leu
Thr Gly Phe Phe Asp Tyr1 5 107511PRTARTIFICIAL SEQUENCECDR-L1 75Arg
Met Ser Gln Asp Ile Ser Ser Tyr Leu Ala1 5 10766PRTARTIFICIAL
SEQUENCECDR-L1 76Gln Asp Ile Ser Ser Tyr1 5777PRTARTIFICIAL
SEQUENCECDR-L2 77Ala Ala Ser Ser Leu Gln Ser1 5783PRTARTIFICIAL
SEQUENCECDR-L2 78Ala Ala Ser1799PRTARTIFICIAL SEQUENCECDR-L3 79Gln
Gln Tyr Asp Ser Phe Pro Pro Thr1 5809PRTARTIFICIAL SEQUENCECDR-H1
80Gly Gly Ser Ile Asn Ser Thr Thr Ser1 58112PRTARTIFICIAL
SEQUENCECDR-H1 81Gly Gly Ser Ile Asn Ser Thr Thr Ser Tyr Trp Ala1 5
10827PRTARTIFICIAL SEQUENCECDR-H1 82Ser Thr Thr Ser Tyr Trp Ala1
58310PRTARTIFICIAL SEQUENCECDR-H1 83Gly Gly Ser Ile Asn Ser Thr Thr
Ser Tyr1 5 10844PRTARTIFICIAL SEQUENCECDR-H2 84Tyr Ser Gly
Lys1859PRTARTIFICIAL SEQUENCECDR-H2 85Thr Ile Phe Tyr Ser Gly Lys
Thr Tyr1 58616PRTARTIFICIAL SEQUENCECDR-H2 86Thr Ile Phe Tyr Ser
Gly Lys Thr Tyr Asn Asn Pro Ser Leu Lys Ser1 5 10
15877PRTARTIFICIAL SEQUENCECDR-H2 87Ile Phe Tyr Ser Gly Lys Thr1
58811PRTARTIFICIAL SEQUENCECDR-H3 88Phe Asp Tyr Gly Phe His Asp Ala
Phe Asp Ile1 5 108913PRTARTIFICIAL SEQUENCECDR-H3 89Ala Arg Phe Asp
Tyr Gly Phe His Asp Ala Phe Asp Ile1 5 109012PRTARTIFICIAL
SEQUENCECDR-L1 90Arg Ala Ser Gln Ser Ile Thr Ser Asp Tyr Leu Ser1 5
10917PRTARTIFICIAL SEQUENCECDR-L1 91Gln Ser Ile Thr Ser Asp Tyr1
5927PRTARTIFICIAL SEQUENCECDR-L2 92Gly Ala Ser Thr Arg Ala Thr1
5933PRTARTIFICIAL SEQUENCECDR-L2 93Gly Ala Ser1948PRTARTIFICIAL
SEQUENCECDR-L3 94Gln Gln Asp Tyr Asn Leu Tyr Thr1 5956PRTARTIFICIAL
SEQUENCECDR-H2 95Ser Ala Tyr Thr Gly Asn1 59610PRTARTIFICIAL
SEQUENCECDR-H2 96Trp Ile Ser Ala Tyr Thr Gly Asn Thr Arg1 5
109717PRTARTIFICIAL SEQUENCECDR-H2 97Trp Ile Ser Ala Tyr Thr Gly
Asn Thr Arg Tyr Ala Gln Lys Leu Gln1 5 10 15Gly988PRTARTIFICIAL
SEQUENCECDR-H2 98Ile Ser Ala Tyr Thr Gly Asn Thr1
59915PRTARTIFICIAL SEQUENCECDR-H3 99Glu Glu Gly Ala Thr Thr Asp Tyr
Asp Tyr Tyr Gly Met Asp Val1 5 10 1510017PRTARTIFICIAL
SEQUENCECDR-H3 100Ala Arg Glu Glu Gly Ala Thr Thr Asp Tyr Asp Tyr
Tyr Gly Met Asp1 5 10 15Val101372DNAARTIFICIAL SEQUENCEVH
101caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggtta cacctttacc agctatggta tcagctgggt
gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atcagcgctt
acaatggtaa cacaaagtat 180gcacagaagc tccagggcag agtcaccatg
accacagaca catccacgag cacagcctac 240atggagctga ggagcctgag
atctgacgac acggccgtgt attactgtgc gagagatgag 300gatattttga
ctggttacaa ctactacggt atggacgtct ggggccaagg gaccacggtc
360accgtctcct ca 372102372DNAARTIFICIAL SEQUENCEVH O/SSE
102caggtgcagc tggttcaatc tggcgccgaa gtgaagaaac caggcgcctc
tgtgaaggtg 60tcctgcaagg ccagcggcta cacctttacc agctacggca tcagctgggt
ccgacaggct 120cctggacaag gcttggaatg gatgggctgg atcagcgcct
acaacggcaa caccaaatac 180gcccagaaac tgcagggcag agtgaccatg
accaccgaca ccagcacaag caccgcctac 240atggaactgc ggagcctgag
atccgatgac accgccgtgt actactgcgc cagagatgag 300gacatcctga
ccggctacaa ctactacggc atggacgtgt ggggccaggg cacaacagtg
360acagtttctt ct 372103124PRTARTIFICIAL
SEQUENCEVH 103Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr
Lys Tyr Ala Gln Lys Leu 50 55 60Gln Gly Arg Val Thr Met Thr Thr Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Glu Asp Ile
Leu Thr Gly Tyr Asn Tyr Tyr Gly Met Asp 100 105 110Val Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser 115 120104336DNAARTIFICIAL
SEQUENCEVL 104cagcttgtgc tgactcaatc gccctctgcc tctgcctccc
tgggagcctc ggtcaagctc 60acctgcactc tgagcagtgg gcacagcagc tacgccatcg
catggcatca gcagcagcca 120gagaagggcc ctcggtactt gatgaagctt
aacagtgatg gcagccacag caagggggac 180gggatccctg atcgcttctc
aggctccagc tctggggctg agcgctacct caccatctcc 240agcctccagt
ctgaggatga ggctgactat tactgtcaga cctggggcac tggcattcgg
300gtgttcggtg gaggaaccaa actgactgtc ctaggc 336105336DNAARTIFICIAL
SEQUENCEVL O/SSE 105caactggtgc tgacacagtc tcctagcgcc tctgcttctc
tgggagccag cgtgaagctg 60acctgtacac tgtctagcgg ccacagcagc tacgccattg
cttggcatca gcagcagccc 120gagaagggcc ctagatacct gatgaagctg
aacagcgacg gcagccactc taaaggcgac 180ggcatccccg atagattcag
cggcagttct agcggagccg agcgctacct gacaatcagc 240tctctgcaat
ccgaggacga ggccgactac tactgtcaga catggggcac cggcatcaga
300gtgtttggcg gaggcaccaa gctgacagtg cttgga 336106112PRTARTIFICIAL
SEQUENCEVL 106Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser
Leu Gly Ala1 5 10 15Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gly His
Ser Ser Tyr Ala 20 25 30Ile Ala Trp His Gln Gln Gln Pro Glu Lys Gly
Pro Arg Tyr Leu Met 35 40 45Lys Leu Asn Ser Asp Gly Ser His Ser Lys
Gly Asp Gly Ile Pro Asp 50 55 60Arg Phe Ser Gly Ser Ser Ser Gly Ala
Glu Arg Tyr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Gln Thr Trp Gly 85 90 95Thr Gly Ile Arg Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105
110107753DNAARTIFICIAL SEQUENCEscFv (VH-VL) 107caggttcagc
tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggtta cacctttacc agctatggta tcagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaatggtaa
cacaaagtat 180gcacagaagc tccagggcag agtcaccatg accacagaca
catccacgag cacagcctac 240atggagctga ggagcctgag atctgacgac
acggccgtgt attactgtgc gagagatgag 300gatattttga ctggttacaa
ctactacggt atggacgtct ggggccaagg gaccacggtc 360accgtctcct
caggtggtgg tggtagcggc ggcggcggct ctggtggtgg tggatcccag
420cttgtgctga ctcaatcgcc ctctgcctct gcctccctgg gagcctcggt
caagctcacc 480tgcactctga gcagtgggca cagcagctac gccatcgcat
ggcatcagca gcagccagag 540aagggccctc ggtacttgat gaagcttaac
agtgatggca gccacagcaa gggggacggg 600atccctgatc gcttctcagg
ctccagctct ggggctgagc gctacctcac catctccagc 660ctccagtctg
aggatgaggc tgactattac tgtcagacct ggggcactgg cattcgggtg
720ttcggtggag gaaccaaact gactgtccta ggc 753108753DNAARTIFICIAL
SEQUENCEscFv (VH-VL) O/SSE 108caggtgcagc tggttcaatc tggcgccgaa
gtgaagaaac caggcgcctc tgtgaaggtg 60tcctgcaagg ccagcggcta cacctttacc
agctacggca tcagctgggt ccgacaggct 120cctggacaag gcttggaatg
gatgggctgg atcagcgcct acaacggcaa caccaaatac 180gcccagaaac
tgcagggcag agtgaccatg accaccgaca ccagcacaag caccgcctac
240atggaactgc ggagcctgag atccgatgac accgccgtgt actactgcgc
cagagatgag 300gacatcctga ccggctacaa ctactacggc atggacgtgt
ggggccaggg cacaacagtg 360acagtttctt ctggcggcgg aggatctggc
ggaggtggaa gcggaggcgg tggatctcaa 420ctggtgctga cacagtctcc
tagcgcctct gcttctctgg gagccagcgt gaagctgacc 480tgtacactgt
ctagcggcca cagcagctac gccattgctt ggcatcagca gcagcccgag
540aagggcccta gatacctgat gaagctgaac agcgacggca gccactctaa
aggcgacggc 600atccccgata gattcagcgg cagttctagc ggagccgagc
gctacctgac aatcagctct 660ctgcaatccg aggacgaggc cgactactac
tgtcagacat ggggcaccgg catcagagtg 720tttggcggag gcaccaagct
gacagtgctt gga 753109251PRTARTIFICIAL SEQUENCEscFv (VH-VL) 109Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Lys Tyr Ala Gln
Lys Leu 50 55 60Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Glu Asp Ile Leu Thr Gly Tyr
Asn Tyr Tyr Gly Met Asp 100 105 110Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser Ser Gly Gly Gly Gly 115 120 125Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gln Leu Val Leu Thr 130 135 140Gln Ser Pro Ser
Ala Ser Ala Ser Leu Gly Ala Ser Val Lys Leu Thr145 150 155 160Cys
Thr Leu Ser Ser Gly His Ser Ser Tyr Ala Ile Ala Trp His Gln 165 170
175Gln Gln Pro Glu Lys Gly Pro Arg Tyr Leu Met Lys Leu Asn Ser Asp
180 185 190Gly Ser His Ser Lys Gly Asp Gly Ile Pro Asp Arg Phe Ser
Gly Ser 195 200 205Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser
Leu Gln Ser Glu 210 215 220Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp
Gly Thr Gly Ile Arg Val225 230 235 240Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 245 250110357DNAARTIFICIAL SEQUENCEVH 110caggtgcagc
tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg
tctctggagg ctccatcagt aattactact ggagctggat ccggcagccc
120gccgggaagg gactggagtg gattgggcgt atctatacca gtgggagcac
caactacaac 180ccctccctca agagtcgagt caccatgtca gtagacacgt
ccaagaacca gttctccctg 240aagctgagtt ctttgaccgc cgcggacacg
gccatatatt actgtgcgag gtattacgat 300attttgactg gtttctttga
ctactggggc cagggaaccc tggtcaccgt ctcctca 357111357DNAARTIFICIAL
SEQUENCEVH O/SSE 111caggttcagc tgcaagagtc tggccctggc ctggtcaagc
ctagcgaaac actgagcctg 60acctgtaccg tgtctggcgg cagcatctcc aactactact
ggtcctggat cagacagcct 120gccggcaaag gcctggaatg gatcggcaga
atctacacca gcggcagcac caactacaac 180cccagcctga agtccagagt
gaccatgagc gtggacacca gcaagaacca gttctccctg 240aagctgagca
gcctgacagc cgccgatacc gccatctact actgtgcccg gtactacgat
300atcctgaccg gcttcttcga ctactggggc cagggaacac tggtcacagt ttctagc
357112119PRTARTIFICIAL SEQUENCEVH 112Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Gly Ser Ile Ser Asn Tyr 20 25 30Tyr Trp Ser Trp Ile
Arg Gln Pro Ala Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Tyr
Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val
Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys
Leu Ser Ser Leu Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90
95Arg Tyr Tyr Asp Ile Leu Thr Gly Phe Phe Asp Tyr Trp Gly Gln Gly
100 105 110Thr Leu Val Thr Val Ser Ser 115113324DNAARTIFICIAL
SEQUENCEVL 113gtcatctgga tgacccagtc tccatcctta ctctctgcat
ctacaggaga cagtgtcacc 60atcagttgtc ggatgagtca ggacattagc agttatttag
cctggtatca gcaaaaacca 120gggaaagccc ctgagctcct gatctatgct
gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc
tgggacagac ttcactctca ccatcagttc cctgcagtct 240gaagattttg
ctacttatta ctgtcaacag tatgatagtt tccctccgac gttcggccaa
300gggaccaagg tggaattcaa acgg 324114324DNAARTIFICIAL SEQUENCEVL
O/SSE 114gtgatttgga tgacacagag ccctagcctg ctgagcgcca gcacaggcga
tagcgtgacc 60atcagctgca gaatgagcca ggacatcagc agctacctgg cttggtatca
gcagaagcct 120ggcaaggccc ctgaactgct gatctatgcc gcttccagtc
tgcagagcgg cgtgccatct 180agattttccg gcagcggctc tggcaccgac
ttcaccctga caatcagctc cctgcagtcc 240gaggacttcg ccacctacta
ttgccagcag tacgacagct tccctccaac ctttggccag 300ggcaccaagg
tggaattcaa gcgc 324115108PRTARTIFICIAL SEQUENCEVL 115Val Ile Trp
Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Thr Gly1 5 10 15Asp Ser
Val Thr Ile Ser Cys Arg Met Ser Gln Asp Ile Ser Ser Tyr 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40
45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Ser65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser
Phe Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Phe Lys Arg
100 105116726DNAARTIFICIAL SEQUENCEscFv (VH-VL) 116caggtgcagc
tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg
tctctggagg ctccatcagt aattactact ggagctggat ccggcagccc
120gccgggaagg gactggagtg gattgggcgt atctatacca gtgggagcac
caactacaac 180ccctccctca agagtcgagt caccatgtca gtagacacgt
ccaagaacca gttctccctg 240aagctgagtt ctttgaccgc cgcggacacg
gccatatatt actgtgcgag gtattacgat 300attttgactg gtttctttga
ctactggggc cagggaaccc tggtcaccgt ctcctcaggt 360ggtggtggta
gcggcggcgg cggctctggt ggtggtggat ccgtcatctg gatgacccag
420tctccatcct tactctctgc atctacagga gacagtgtca ccatcagttg
tcggatgagt 480caggacatta gcagttattt agcctggtat cagcaaaaac
cagggaaagc ccctgagctc 540ctgatctatg ctgcatccag tttgcaaagt
ggggtcccat caaggttcag tggcagtgga 600tctgggacag acttcactct
caccatcagt tccctgcagt ctgaagattt tgctacttat 660tactgtcaac
agtatgatag tttccctccg acgttcggcc aagggaccaa ggtggaattc 720aaacgg
726117726DNAARTIFICIAL SEQUENCEscFv (VH-VL) O/SSE 117caggttcagc
tgcaagagtc tggccctggc ctggtcaagc ctagcgaaac actgagcctg 60acctgtaccg
tgtctggcgg cagcatctcc aactactact ggtcctggat cagacagcct
120gccggcaaag gcctggaatg gatcggcaga atctacacca gcggcagcac
caactacaac 180cccagcctga agtccagagt gaccatgagc gtggacacca
gcaagaacca gttctccctg 240aagctgagca gcctgacagc cgccgatacc
gccatctact actgtgcccg gtactacgat 300atcctgaccg gcttcttcga
ctactggggc cagggaacac tggtcacagt ttctagcgga 360ggcggaggat
ctggtggcgg aggaagtggc ggaggcggtt ctgtgatttg gatgacacag
420agccctagcc tgctgagcgc cagcacaggc gatagcgtga ccatcagctg
cagaatgagc 480caggacatca gcagctacct ggcttggtat cagcagaagc
ctggcaaggc ccctgaactg 540ctgatctatg ccgcttccag tctgcagagc
ggcgtgccat ctagattttc cggcagcggc 600tctggcaccg acttcaccct
gacaatcagc tccctgcagt ccgaggactt cgccacctac 660tattgccagc
agtacgacag cttccctcca acctttggcc agggcaccaa ggtggaattc 720aagcgc
726118242PRTARTIFICIAL SEQUENCEscFv (VH-VL) 118Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Tyr 20 25 30Tyr Trp Ser
Trp Ile Arg Gln Pro Ala Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Arg
Ile Tyr Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser
Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Leu Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95Arg Tyr Tyr Asp Ile Leu Thr Gly Phe Phe Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly 115 120 125Ser Gly Gly Gly Gly Ser Val Ile Trp Met Thr
Gln Ser Pro Ser Leu 130 135 140Leu Ser Ala Ser Thr Gly Asp Ser Val
Thr Ile Ser Cys Arg Met Ser145 150 155 160Gln Asp Ile Ser Ser Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175Ala Pro Glu Leu
Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val 180 185 190Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200
205Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
210 215 220Tyr Asp Ser Phe Pro Pro Thr Phe Gly Gln Gly Thr Lys Val
Glu Phe225 230 235 240Lys Arg119363DNAARTIFICIAL SEQUENCEVH
119cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac
cctgtccctc 60acctgcactg tctctggtgg ctccatcaac agtactactt cctactgggc
ctggatccgc 120cagcccccag ggaaggggct ggagtggatt gggactatct
tttatagtgg gaaaacctac 180aacaacccgt ccctcaagag tcgagtcacc
atgtccgtag acacgtccaa gaaccacttc 240tccctgaagg tgaactctgt
gaccgccgca gacacggctg tgtattactg tgcgaggttt 300gactacggtt
ttcatgatgc ttttgatatc tggggccagg ggacaatggt caccgtctct 360tca
363120363DNAARTIFICIAL SEQUENCEVH O/SSE 120cagctccagc tgcaagaatc
tggacctggc ctggtcaagc ccagcgagac actgtctctg 60acctgtacag tgtccggcgg
cagcatcaat agcaccacaa gctactgggc ctggatcaga 120cagcctcctg
gcaaaggcct ggaatggatc ggcaccatct tctacagcgg caagacctac
180aacaacccca gcctgaagtc cagagtgacc atgagcgtgg acaccagcaa
gaaccacttc 240agcctgaaag tgaacagcgt gacagccgcc gataccgccg
tgtactactg cgccagattc 300gactacggct tccacgacgc cttcgacatc
tggggccagg gcacaatggt cacagtttct 360agc 363121121PRTARTIFICIAL
SEQUENCEVH 121Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser
Ile Asn Ser Thr 20 25 30Thr Ser Tyr Trp Ala Trp Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu 35 40 45Trp Ile Gly Thr Ile Phe Tyr Ser Gly Lys
Thr Tyr Asn Asn Pro Ser 50 55 60Leu Lys Ser Arg Val Thr Met Ser Val
Asp Thr Ser Lys Asn His Phe65 70 75 80Ser Leu Lys Val Asn Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg Phe Asp Tyr
Gly Phe His Asp Ala Phe Asp Ile Trp Gly 100 105 110Gln Gly Thr Met
Val Thr Val Ser Ser 115 120122324DNAARTIFICIAL SEQUENCEVL
122gaaattgtaa tgacacagtc tccagccacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtattacc agcgactact tatcctggta
ccaacaaaaa 120cctgggcagg ctcccaggct cctcatctat ggtgcatcca
ccagggccac tggcatccca 180gccaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagcctgcag 240cctgaagatt ttgtagttta
ttactgtcag caggattata acttgtacac ttttggccag 300gggaccaagc
tggagatcaa acgg 324123324DNAARTIFICIAL SEQUENCEVL O/SSE
123gagattgtga tgacacagag ccccgccact ctgagcctta gtcctggcga
aagagccaca 60ctgagctgca gagccagcca gagcatcacc agcgattacc tgagctggta
tcagcagaag 120cccggacagg ctcccagact gctgatctat ggcgcctcta
caagagccac cggcattccc 180gcccgctttt ctggctctgg aagcggcacc
gacttcaccc tgaccatatc tagcctgcag 240cctgaggact tcgtggtgta
ctattgccag caggactaca acctgtacac cttcggccag 300gggaccaagc
tggaaatcaa gaga 324124108PRTARTIFICIAL SEQUENCEVL 124Glu Ile Val
Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Thr Ser Asp 20 25 30Tyr
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40
45Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln65 70 75 80Pro Glu Asp Phe Val Val Tyr Tyr Cys Gln Gln Asp Tyr
Asn Leu Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105125732DNAARTIFICIAL SEQUENCEscFv (VH-VL) 125cagctgcagc
tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg
tctctggtgg ctccatcaac agtactactt cctactgggc ctggatccgc
120cagcccccag ggaaggggct ggagtggatt gggactatct tttatagtgg
gaaaacctac 180aacaacccgt ccctcaagag tcgagtcacc atgtccgtag
acacgtccaa gaaccacttc 240tccctgaagg tgaactctgt
gaccgccgca gacacggctg tgtattactg tgcgaggttt 300gactacggtt
ttcatgatgc ttttgatatc tggggccagg ggacaatggt caccgtctct
360tcaggtggtg gtggtagcgg cggcggcggc tctggtggtg gtggatccga
aattgtaatg 420acacagtctc cagccaccct gtctttgtct ccaggggaaa
gagccaccct ctcctgcagg 480gccagtcaga gtattaccag cgactactta
tcctggtacc aacaaaaacc tgggcaggct 540cccaggctcc tcatctatgg
tgcatccacc agggccactg gcatcccagc caggttcagt 600ggcagtgggt
ctgggacaga cttcactctc accatcagca gcctgcagcc tgaagatttt
660gtagtttatt actgtcagca ggattataac ttgtacactt ttggccaggg
gaccaagctg 720gagatcaaac gg 732126732DNAARTIFICIAL SEQUENCEscFv
(VH-VL) O/SSE 126cagctccagc tgcaagaatc tggacctggc ctggtcaagc
ccagcgagac actgtctctg 60acctgtacag tgtccggcgg cagcatcaat agcaccacaa
gctactgggc ctggatcaga 120cagcctcctg gcaaaggcct ggaatggatc
ggcaccatct tctacagcgg caagacctac 180aacaacccca gcctgaagtc
cagagtgacc atgagcgtgg acaccagcaa gaaccacttc 240agcctgaaag
tgaacagcgt gacagccgcc gataccgccg tgtactactg cgccagattc
300gactacggct tccacgacgc cttcgacatc tggggccagg gcacaatggt
cacagtttct 360agcggaggcg gaggatctgg tggcggagga agtggcggag
gcggttctga gattgtgatg 420acacagagcc ccgccactct gagccttagt
cctggcgaaa gagccacact gagctgcaga 480gccagccaga gcatcaccag
cgattacctg agctggtatc agcagaagcc cggacaggct 540cccagactgc
tgatctatgg cgcctctaca agagccaccg gcattcccgc ccgcttttct
600ggctctggaa gcggcaccga cttcaccctg accatatcta gcctgcagcc
tgaggacttc 660gtggtgtact attgccagca ggactacaac ctgtacacct
tcggccaggg gaccaagctg 720gaaatcaaga ga 732127244PRTARTIFICIAL
SEQUENCEscFv (VH-VL) 127Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
Gly Ser Ile Asn Ser Thr 20 25 30Thr Ser Tyr Trp Ala Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu 35 40 45Trp Ile Gly Thr Ile Phe Tyr Ser
Gly Lys Thr Tyr Asn Asn Pro Ser 50 55 60Leu Lys Ser Arg Val Thr Met
Ser Val Asp Thr Ser Lys Asn His Phe65 70 75 80Ser Leu Lys Val Asn
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg Phe
Asp Tyr Gly Phe His Asp Ala Phe Asp Ile Trp Gly 100 105 110Gln Gly
Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120
125Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro
130 135 140Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser
Cys Arg145 150 155 160Ala Ser Gln Ser Ile Thr Ser Asp Tyr Leu Ser
Trp Tyr Gln Gln Lys 165 170 175Pro Gly Gln Ala Pro Arg Leu Leu Ile
Tyr Gly Ala Ser Thr Arg Ala 180 185 190Thr Gly Ile Pro Ala Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205Thr Leu Thr Ile Ser
Ser Leu Gln Pro Glu Asp Phe Val Val Tyr Tyr 210 215 220Cys Gln Gln
Asp Tyr Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu225 230 235
240Glu Ile Lys Arg128372DNAARTIFICIAL SEQUENCEVH 128caggttcagc
tgctgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggtta cacctttacc agctatggta tcagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcagcgctt acactggtaa
cacaaggtat 180gcacagaagc tccagggcag agtcaccatg accacagaca
catccacgag cacagcctac 240atggagctga ggagcctgag atctgacgac
acggccgtgt attactgtgc gagagaagaa 300ggagctacta cggactacga
ctactacggt atggacgtct ggggccaagg gactgcggtc 360accgtctcct ca
372129372DNAARTIFICIAL SEQUENCEVH O/SSE 129caggttcagc tgcttcagtc
tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60tcctgcaagg ccagcggcta
cacctttacc agctacggca tcagctgggt ccgacaggct 120cctggacaag
gcttggaatg gatgggctgg atcagcgcct acaccggcaa taccagatac
180gcccagaaac tgcagggcag agtgaccatg accaccgaca ccagcacaag
caccgcctac 240atggaactgc ggagcctgag atccgatgac accgccgtgt
actactgcgc cagagaagaa 300ggcgccacca ccgactacga ctactacggc
atggatgtgt ggggccaggg aacagccgtg 360acagtttctt ct
372130124PRTARTIFICIAL SEQUENCEVH 130Gln Val Gln Leu Leu Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Gly Ile Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Ser
Ala Tyr Thr Gly Asn Thr Arg Tyr Ala Gln Lys Leu 50 55 60Gln Gly Arg
Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu Glu Gly Ala Thr Thr Asp Tyr Asp Tyr Tyr Gly Met Asp
100 105 110Val Trp Gly Gln Gly Thr Ala Val Thr Val Ser Ser 115
120131336DNAartificial sequenceVL O/SSE 131caactggttc tgacacagag
cccaagcgcc tctgcatctc tgggagcttc cgtgaagctg 60acctgcacac tgtctagcgg
ccacagcagc tatgccattg cctggcatca gcaacagccc 120gagaagggcc
ctagatacct gatgaagctg aacagcgacg gcagccactc taaaggcgac
180ggcatccccg atagattcag cggcagttct agcggagccg agcgctacct
gacaatcagc 240tctctgcaat ccgaggacga ggccgattac tactgtcaga
catggggcac cggcatcaga 300gtgtttggcg gcggaacaaa gctgaccgtg ctgggc
336132696DNAartificial sequencescFv (VH-VL) 132caggttcagc
tgctgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggtta cacctttacc agctatggta tcagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcagcgctt acactggtaa
cacaaggtat 180gcacagaagc tccagggcag agtcaccatg accacagaca
catccacgag cacagcctac 240atggagctga ggagcctgag atctgacgac
acggccgtgt attactgtgc gagagaagaa 300ggagctacta cggactacga
ctactacggt atggacgtct ggggccaagg gactgcggtc 360accgtctcct
caggtggtgg tggtagcggc ggcggcggct ctggtggtgg tggatcccag
420cttgtgctga ctcaatcgcc ctctgcctct gcctccctgg gagcctcggt
caagctcacc 480tgcactctga gcagtgggca cagcagctac gccatcgcat
ggcatcagca gcagccagag 540aagggccctc ggtacttgat gaagcttaac
agtgatggca gccacagcaa gggggacggg 600atccctgatc gcttctcagg
ctccagctct ggggctgagc gctacctcac catctccagc 660ctccagtctg
aggatgaggc tgactattac tgtcag 696133753DNAartificial sequencescFv
(VH-VL) O/SSE 133caggttcagc tgcttcagtc tggcgccgaa gtgaagaaac
ctggcgcctc tgtgaaggtg 60tcctgcaagg ccagcggcta cacctttacc agctacggca
tcagctgggt ccgacaggct 120cctggacaag gcttggaatg gatgggctgg
atcagcgcct acaccggcaa taccagatac 180gcccagaaac tgcagggcag
agtgaccatg accaccgaca ccagcacaag caccgcctac 240atggaactgc
ggagcctgag atccgatgac accgccgtgt actactgcgc cagagaagaa
300ggcgccacca ccgactacga ctactacggc atggatgtgt ggggccaggg
aacagccgtg 360acagtttctt ctggtggcgg aggatctggc ggaggtggaa
gcggcggagg cggatctcaa 420ctggttctga cacagagccc aagcgcctct
gcatctctgg gagcttccgt gaagctgacc 480tgcacactgt ctagcggcca
cagcagctat gccattgcct ggcatcagca acagcccgag 540aagggcccta
gatacctgat gaagctgaac agcgacggca gccactctaa aggcgacggc
600atccccgata gattcagcgg cagttctagc ggagccgagc gctacctgac
aatcagctct 660ctgcaatccg aggacgaggc cgattactac tgtcagacat
ggggcaccgg catcagagtg 720tttggcggcg gaacaaagct gaccgtgctg ggc
753134251PRTartificial sequencescFv (VH-VL) 134Gln Val Gln Leu Leu
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Gly Ile Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Ser Ala Tyr Thr Gly Asn Thr Arg Tyr Ala Gln Lys Leu 50 55 60Gln
Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Glu Gly Ala Thr Thr Asp Tyr Asp Tyr Tyr Gly Met
Asp 100 105 110Val Trp Gly Gln Gly Thr Ala Val Thr Val Ser Ser Gly
Gly Gly Gly 115 120 125Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gln Leu Val Leu Thr 130 135 140Gln Ser Pro Ser Ala Ser Ala Ser Leu
Gly Ala Ser Val Lys Leu Thr145 150 155 160Cys Thr Leu Ser Ser Gly
His Ser Ser Tyr Ala Ile Ala Trp His Gln 165 170 175Gln Gln Pro Glu
Lys Gly Pro Arg Tyr Leu Met Lys Leu Asn Ser Asp 180 185 190Gly Ser
His Ser Lys Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser 195 200
205Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu
210 215 220Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Gly Thr Gly Ile
Arg Val225 230 235 240Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
245 25013513PRTartificial sequenceIgG4 hinge spacer 135Glu Ser Lys
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met1 5 1013639DNAartificial
sequenceIgG4 hinge spacer O/SSE 136gagtctaaat acggaccgcc ttgtcctcct
tgtcccatg 39137357DNAartificial sequenceHinge-CH3 spacer O/SSE
137gagtctaaat acggaccgcc ttgtcctcct tgtcccggcc agccaagaga
gccccaggtt 60tacacactgc ctccaagcca agaggaaatg accaagaatc aggtgtccct
gacatgcctg 120gtcaagggct tctacccctc cgatatcgcc gtggaatggg
agagcaatgg ccagcctgag 180aacaactaca agaccacacc tcctgtgctg
gacagcgacg gcagtttctt cctgtatagt 240agactcaccg tggataaatc
aagatggcaa gagggcaacg tgttcagctg cagcgtgatg 300cacgaggccc
tgcacaacca ctacacccag aaaagcctga gcctgtctct gggcaag
357138120PRTartificial sequenceHinge-CH3 spacer 138Glu Ser Lys Tyr
Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg1 5 10 15Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 20 25 30Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 35 40 45Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 50 55
60Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser65
70 75 80Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
Ser 85 90 95Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser 100 105 110Leu Ser Leu Ser Leu Gly Lys Met 115
120139360DNAartificial sequenceHinge-CH3 spacer O/SSE 139gagtctaaat
acggaccgcc ttgtcctcct tgtcccggcc agccaagaga gccccaggtt 60tacacactgc
ctccaagcca agaggaaatg accaagaatc aggtgtccct gacatgcctg
120gtcaagggct tctacccctc cgatatcgcc gtggaatggg agagcaatgg
ccagcctgag 180aacaactaca agaccacacc tcctgtgctg gacagcgacg
gcagtttctt cctgtatagt 240agactcaccg tggataaatc aagatggcaa
gagggcaacg tgttcagctg cagcgtgatg 300cacgaggccc tgcacaacca
ctacacccag aaaagcctga gcctgtctct gggcaagatg 360140684DNAartificial
sequenceIgG4/IgG2 hinge- IgG2/IgG4 CH2- IgG4 CH3 spacer O/SSE
140gagtctaaat acggaccgcc ttgtcctcct tgtcccgctc ctcctgttgc
cggaccttcc 60gtgttcctgt ttcctccaaa gcctaaggac accctgatga tcagcaggac
ccctgaagtg 120acctgcgtgg tggtggatgt gtcccaagag gatcccgagg
tgcagttcaa ctggtatgtg 180gacggcgtgg aagtgcacaa cgccaagacc
aagcctagag aggaacagtt ccagagcacc 240tacagagtgg tgtccgtgct
gacagtgctg caccaggatt ggctgaacgg caaagagtac 300aagtgcaagg
tgtccaacaa gggcctgcct agcagcatcg agaaaaccat ctccaaggcc
360aagggccagc caagagagcc ccaggtttac acactgcctc caagccaaga
ggaaatgacc 420aagaatcagg tgtccctgac atgcctggtc aagggcttct
acccctccga tatcgccgtg 480gaatgggaga gcaatggcca gcctgagaac
aactacaaga ccacacctcc tgtgctggac 540agcgacggca gtttcttcct
gtatagtaga ctcaccgtgg ataaatcaag atggcaagag 600ggcaacgtgt
tcagctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaaa
660agcctgagcc tgtctctggg caag 68414172DNAartificial sequenceT2A
O/SSE 141cttgaaggtg gtggcgaagg cagaggcagc ctgcttacat gcggagatgt
ggaagagaac 60cccggaccta ga 72142248PRTartificial sequenceR12 VH-VL
scFv 142Gln Glu Gln Leu Val Glu Ser Gly Gly Arg Leu Val Thr Pro Gly
Gly1 5 10 15Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly Phe Asp Phe Ser
Ala Tyr 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr Tyr Tyr
Ala Thr Trp Val 50 55 60Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala
Gln Asn Thr Val Asp65 70 75 80Leu Gln Met Asn Ser Leu Thr Ala Ala
Asp Arg Ala Thr Tyr Phe Cys 85 90 95Ala Arg Asp Ser Tyr Ala Asp Asp
Gly Ala Leu Phe Asn Ile Trp Gly 100 105 110Pro Gly Thr Leu Val Thr
Ile Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly Ser Gly
Gly Gly Gly Ser Glu Leu Val Leu Thr Gln Ser Pro 130 135 140Ser Val
Ser Ala Ala Leu Gly Ser Pro Ala Lys Ile Thr Cys Thr Leu145 150 155
160Ser Ser Ala His Lys Thr Asp Thr Ile Asp Trp Tyr Gln Gln Leu Gln
165 170 175Gly Glu Ala Pro Arg Tyr Leu Met Gln Val Gln Ser Asp Gly
Ser Tyr 180 185 190Thr Lys Arg Pro Gly Val Pro Asp Arg Phe Ser Gly
Ser Ser Ser Gly 195 200 205Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
Gln Ala Asp Asp Glu Ala 210 215 220Asp Tyr Tyr Cys Gly Ala Asp Tyr
Ile Gly Gly Tyr Val Phe Gly Gly225 230 235 240Gly Thr Gln Leu Thr
Val Thr Gly 245143745DNAartificial sequenceR12 VH-VL scFv
143caagaacagc tggtggaatc tggcggcaga ctggttacac ctggcggaag
cctgacactg 60agctgtaaag ccagcggctt cgacttcagc gcctactaca tgagctgggt
ccgacaggcc 120cctggcaaag gactggaatg gatcgccaca atctacccca
gctccggcaa gacctactac 180gccacatggg tcaacggccg gttcaccatc
agcagcgaca acgcccagaa caccgtggac 240ctgcagatga actctctgac
agccgccgac cgggccacct acttttgtgc cagagatagc 300tacgccgacg
acggcgccct gttcaatatt tggggacctg gcacactcgt gaccatctct
360agcggaggcg gaggaagtgg tggcggagga tcaggcggtg gtggatctga
actggtgctg 420acacagagcc cctctgtgtc tgctgctctg ggaagccctg
ccaagatcac atgtaccctg 480agcagcgccc acaagaccga caccatcgac
tggtatcagc agctgcaggg cgaagcccct 540agatacctga tgcaggttca
gagcgacggc agctacacca aaagacctgg cgtgcccgat 600agattcagcg
gcagttcttc tggcgccgat cgctacctga tcatcccttc tgtgcaagcc
660gacgatgagg ccgactatta ctgcggagcc gattacatcg gcggctacgt
tttcggtggc 720ggcacacagt tgacagtgac aggcg 745144908PRThomo
sapiensMISC_FEATUREhuman ROR1 GenBank AAA60275.1 144Gln Glu Thr Glu
Leu Ser Val Ser Ala Glu Leu Val Pro Thr Ser Ser1 5 10 15Trp Asn Ile
Ser Ser Glu Leu Asn Lys Asp Ser Tyr Leu Thr Leu Asp 20 25 30Glu Pro
Met Asn Asn Ile Thr Thr Ser Leu Gly Gln Thr Ala Glu Leu 35 40 45His
Cys Lys Val Ser Gly Asn Pro Pro Pro Thr Ile Arg Trp Phe Lys 50 55
60Asn Asp Ala Pro Val Val Gln Glu Pro Arg Arg Leu Ser Phe Arg Ser65
70 75 80Thr Ile Tyr Gly Ser Arg Leu Arg Ile Arg Asn Leu Asp Thr Thr
Asp 85 90 95Thr Gly Tyr Phe Gln Cys Val Ala Thr Asn Gly Lys Glu Val
Val Ser 100 105 110Ser Thr Gly Val Leu Phe Val Lys Phe Gly Pro Pro
Pro Thr Ala Ser 115 120 125Pro Gly Tyr Ser Asp Glu Tyr Glu Glu Asp
Gly Phe Cys Gln Pro Tyr 130 135 140Arg Gly Ile Ala Cys Ala Arg Phe
Ile Gly Asn Arg Thr Val Tyr Met145 150 155 160Glu Ser Leu His Met
Gln Gly Glu Ile Glu Asn Gln Ile Thr Ala Ala 165 170 175Phe Thr Met
Ile Gly Thr Ser Ser His Leu Ser Asp Lys Cys Ser Gln 180 185 190Phe
Ala Ile Pro Ser Leu Cys His Tyr Ala Phe Pro Tyr Cys Asp Glu 195 200
205Thr Ser Ser Val Pro Lys Pro Arg Asp Leu Cys Arg Asp Glu Cys Glu
210 215 220Ile Leu Glu Asn Val Leu Cys Gln Thr Glu Tyr Ile Phe Ala
Arg Ser225 230 235 240Asn Pro Met Ile Leu Met Arg Leu Lys Leu Pro
Asn Cys Glu Asp Leu 245 250 255Pro Gln Pro Glu Ser Pro Glu Ala Ala
Asn Cys Ile Arg Ile Gly Ile 260 265 270Pro Met Ala Asp Pro Ile Asn
Lys Asn His Lys Cys Tyr Asn Ser Thr 275
280 285Gly Val Asp Tyr Arg Gly Thr Val Ser Val Thr Lys Ser Gly Arg
Gln 290 295 300Cys Gln Pro Trp Asn Ser Gln Tyr Pro His Thr His Thr
Phe Thr Ala305 310 315 320Leu Arg Phe Pro Glu Leu Asn Gly Gly His
Ser Tyr Cys Arg Asn Pro 325 330 335Gly Asn Gln Lys Glu Ala Pro Trp
Cys Phe Thr Leu Asp Glu Asn Phe 340 345 350Lys Ser Asp Leu Cys Asp
Ile Pro Ala Cys Asp Ser Lys Asp Ser Lys 355 360 365Glu Lys Asn Lys
Met Glu Ile Leu Tyr Ile Leu Val Pro Ser Val Ala 370 375 380Ile Pro
Leu Ala Ile Ala Leu Leu Phe Phe Phe Ile Cys Val Cys Arg385 390 395
400Asn Asn Gln Lys Ser Ser Ser Ala Pro Val Gln Arg Gln Pro Lys His
405 410 415Val Arg Gly Gln Asn Val Glu Met Ser Met Leu Asn Ala Tyr
Lys Pro 420 425 430Lys Ser Lys Ala Lys Glu Leu Pro Leu Ser Ala Val
Arg Phe Met Glu 435 440 445Glu Leu Gly Glu Cys Ala Phe Gly Lys Ile
Tyr Lys Gly His Leu Tyr 450 455 460Leu Pro Gly Met Asp His Ala Gln
Leu Val Ala Ile Lys Thr Leu Lys465 470 475 480Asp Tyr Asn Asn Pro
Gln Gln Trp Thr Glu Phe Gln Gln Glu Ala Ser 485 490 495Leu Met Ala
Glu Leu His His Pro Asn Ile Val Cys Leu Leu Gly Ala 500 505 510Val
Thr Gln Glu Gln Pro Val Cys Met Leu Phe Glu Tyr Ile Asn Gln 515 520
525Gly Asp Leu His Glu Phe Leu Ile Met Arg Ser Pro His Ser Asp Val
530 535 540Gly Cys Ser Ser Asp Glu Asp Gly Thr Val Lys Ser Ser Leu
Asp His545 550 555 560Gly Asp Phe Leu His Ile Ala Ile Gln Ile Ala
Ala Gly Met Glu Tyr 565 570 575Leu Ser Ser His Phe Phe Val His Lys
Asp Leu Ala Ala Arg Asn Ile 580 585 590Leu Ile Gly Glu Gln Leu His
Val Lys Ile Ser Asp Leu Gly Leu Ser 595 600 605Arg Glu Ile Tyr Ser
Ala Asp Tyr Tyr Arg Val Gln Ser Lys Ser Leu 610 615 620Leu Pro Ile
Arg Trp Met Pro Pro Glu Ala Ile Met Tyr Gly Lys Phe625 630 635
640Ser Ser Asp Ser Asp Ile Trp Ser Phe Gly Val Val Leu Trp Glu Ile
645 650 655Phe Ser Phe Gly Leu Gln Pro Tyr Tyr Gly Phe Ser Asn Gln
Glu Val 660 665 670Ile Glu Met Val Arg Lys Arg Gln Leu Leu Pro Cys
Ser Glu Asp Cys 675 680 685Pro Pro Arg Met Tyr Ser Leu Met Thr Glu
Cys Trp Asn Glu Ile Pro 690 695 700Ser Arg Arg Pro Arg Phe Lys Asp
Ile His Val Arg Leu Arg Ser Trp705 710 715 720Glu Gly Leu Ser Ser
His Thr Ser Ser Thr Thr Pro Ser Gly Gly Asn 725 730 735Ala Thr Thr
Gln Thr Thr Ser Leu Ser Ala Ser Pro Val Ser Asn Leu 740 745 750Ser
Asn Pro Arg Tyr Pro Asn Tyr Met Phe Pro Ser Gln Gly Ile Thr 755 760
765Pro Gln Gly Gln Ile Ala Gly Phe Ile Gly Pro Pro Ile Pro Gln Asn
770 775 780Gln Arg Phe Ile Pro Ile Asn Gly Tyr Pro Ile Pro Pro Gly
Tyr Ala785 790 795 800Ala Phe Pro Ala Ala His Tyr Gln Pro Thr Gly
Pro Pro Arg Val Ile 805 810 815Gln His Cys Pro Pro Pro Lys Ser Arg
Ser Pro Ser Ser Ala Ser Gly 820 825 830Ser Thr Ser Thr Gly His Val
Thr Ser Leu Pro Ser Ser Gly Ser Asn 835 840 845Gln Glu Ala Asn Ile
Pro Leu Leu Pro His Met Ser Ile Pro Asn His 850 855 860Pro Gly Gly
Met Gly Ile Thr Val Phe Gly Asn Lys Ser Gln Lys Pro865 870 875
880Tyr Lys Ile Asp Ser Lys Gln Ala Ser Leu Leu Gly Asp Ala Asn Ile
885 890 895His Gly His Thr Glu Ser Met Ile Ser Ala Glu Leu 900
905145520PRThomo sapiensMISC_FEATUREhuman ROR1 isoform 2 145Gln Glu
Thr Glu Leu Ser Val Ser Ala Glu Leu Val Pro Thr Ser Ser1 5 10 15Trp
Asn Ile Ser Ser Glu Leu Asn Lys Asp Ser Tyr Leu Thr Leu Asp 20 25
30Glu Pro Met Asn Asn Ile Thr Thr Ser Leu Gly Gln Thr Ala Glu Leu
35 40 45His Cys Lys Val Ser Gly Asn Pro Pro Pro Thr Ile Arg Trp Phe
Lys 50 55 60Asn Asp Ala Pro Val Val Gln Glu Pro Arg Arg Leu Ser Phe
Arg Ser65 70 75 80Thr Ile Tyr Gly Ser Arg Leu Arg Ile Arg Asn Leu
Asp Thr Thr Asp 85 90 95Thr Gly Tyr Phe Gln Cys Val Ala Thr Asn Gly
Lys Glu Val Val Ser 100 105 110Ser Thr Gly Val Leu Phe Val Lys Phe
Gly Pro Pro Pro Thr Ala Ser 115 120 125Pro Gly Tyr Ser Asp Glu Tyr
Glu Glu Asp Gly Phe Cys Gln Pro Tyr 130 135 140Arg Gly Ile Ala Cys
Ala Arg Phe Ile Gly Asn Arg Thr Val Tyr Met145 150 155 160Glu Ser
Leu His Met Gln Gly Glu Ile Glu Asn Gln Ile Thr Ala Ala 165 170
175Phe Thr Met Ile Gly Thr Ser Ser His Leu Ser Asp Lys Cys Ser Gln
180 185 190Phe Ala Ile Pro Ser Leu Cys His Tyr Ala Phe Pro Tyr Cys
Asp Glu 195 200 205Thr Ser Ser Val Pro Lys Pro Arg Asp Leu Cys Arg
Asp Glu Cys Glu 210 215 220Ile Leu Glu Asn Val Leu Cys Gln Thr Glu
Tyr Ile Phe Ala Arg Ser225 230 235 240Asn Pro Met Ile Leu Met Arg
Leu Lys Leu Pro Asn Cys Glu Asp Leu 245 250 255Pro Gln Pro Glu Ser
Pro Glu Ala Ala Asn Cys Ile Arg Ile Gly Ile 260 265 270Pro Met Ala
Asp Pro Ile Asn Lys Asn His Lys Cys Tyr Asn Ser Thr 275 280 285Gly
Val Asp Tyr Arg Gly Thr Val Ser Val Thr Lys Ser Gly Arg Gln 290 295
300Cys Gln Pro Trp Asn Ser Gln Tyr Pro His Thr His Thr Phe Thr
Ala305 310 315 320Leu Arg Phe Pro Glu Leu Asn Gly Gly His Ser Tyr
Cys Arg Asn Pro 325 330 335Gly Asn Gln Lys Glu Ala Pro Trp Cys Phe
Thr Leu Asp Glu Asn Phe 340 345 350Lys Ser Asp Leu Cys Asp Ile Pro
Ala Cys Asp Ser Lys Asp Ser Lys 355 360 365Glu Lys Asn Lys Met Glu
Ile Leu Tyr Ile Leu Val Pro Ser Val Ala 370 375 380Ile Pro Leu Ala
Ile Ala Leu Leu Phe Phe Phe Ile Cys Val Cys Arg385 390 395 400Asn
Asn Gln Lys Ser Ser Ser Ala Pro Val Gln Arg Gln Pro Lys His 405 410
415Val Arg Gly Gln Asn Val Glu Met Ser Met Leu Asn Ala Tyr Lys Pro
420 425 430Lys Ser Lys Ala Lys Glu Leu Pro Leu Ser Ala Val Arg Phe
Met Glu 435 440 445Glu Leu Gly Glu Cys Ala Phe Gly Lys Ile Tyr Lys
Gly His Leu Tyr 450 455 460Leu Pro Gly Met Asp His Ala Gln Leu Val
Ala Ile Lys Thr Leu Lys465 470 475 480Asp Tyr Asn Asn Pro Gln Gln
Trp Thr Glu Phe Gln Gln Glu Ala Ser 485 490 495Leu Met Ala Glu Leu
His His Pro Asn Ile Val Cys Leu Leu Gly Ala 500 505 510Val Thr Gln
Glu Gln Pro Val Cys 515 520146364PRThomo sapiensMISC_FEATUREhuman
ROR1 isoform 3 146Gln Glu Thr Glu Leu Ser Val Ser Ala Glu Leu Val
Pro Thr Ser Ser1 5 10 15Trp Asn Ile Ser Ser Glu Leu Asn Lys Asp Ser
Tyr Leu Thr Leu Asp 20 25 30Glu Pro Met Asn Asn Ile Thr Thr Ser Leu
Gly Gln Thr Ala Glu Leu 35 40 45His Cys Lys Val Ser Gly Asn Pro Pro
Pro Thr Ile Arg Trp Phe Lys 50 55 60Asn Asp Ala Pro Val Val Gln Glu
Pro Arg Arg Leu Ser Phe Arg Ser65 70 75 80Thr Ile Tyr Gly Ser Arg
Leu Arg Ile Arg Asn Leu Asp Thr Thr Asp 85 90 95Thr Gly Tyr Phe Gln
Cys Val Ala Thr Asn Gly Lys Glu Val Val Ser 100 105 110Ser Thr Gly
Val Leu Phe Val Lys Phe Gly Pro Pro Pro Thr Ala Ser 115 120 125Pro
Gly Tyr Ser Asp Glu Tyr Glu Glu Asp Gly Phe Cys Gln Pro Tyr 130 135
140Arg Gly Ile Ala Cys Ala Arg Phe Ile Gly Asn Arg Thr Val Tyr
Met145 150 155 160Glu Ser Leu His Met Gln Gly Glu Ile Glu Asn Gln
Ile Thr Ala Ala 165 170 175Phe Thr Met Ile Gly Thr Ser Ser His Leu
Ser Asp Lys Cys Ser Gln 180 185 190Phe Ala Ile Pro Ser Leu Cys His
Tyr Ala Phe Pro Tyr Cys Asp Glu 195 200 205Thr Ser Ser Val Pro Lys
Pro Arg Asp Leu Cys Arg Asp Glu Cys Glu 210 215 220Ile Leu Glu Asn
Val Leu Cys Gln Thr Glu Tyr Ile Phe Ala Arg Ser225 230 235 240Asn
Pro Met Ile Leu Met Arg Leu Lys Leu Pro Asn Cys Glu Asp Leu 245 250
255Pro Gln Pro Glu Ser Pro Glu Ala Ala Asn Cys Ile Arg Ile Gly Ile
260 265 270Pro Met Ala Asp Pro Ile Asn Lys Asn His Lys Cys Tyr Asn
Ser Thr 275 280 285Gly Val Asp Tyr Arg Gly Thr Val Ser Val Thr Lys
Ser Gly Arg Gln 290 295 300Cys Gln Pro Trp Asn Ser Gln Tyr Pro His
Thr His Thr Phe Thr Ala305 310 315 320Leu Arg Phe Pro Glu Leu Asn
Gly Gly His Ser Tyr Cys Arg Asn Pro 325 330 335Gly Asn Gln Lys Glu
Ala Pro Trp Cys Phe Thr Leu Asp Glu Asn Phe 340 345 350Lys Ser Asp
Leu Cys Asp Ile Pro Ala Cys Gly Lys 355 36014784DNAartificial
sequenceCD28 transmembrane domain 147atgttttggg tgctggtcgt
ggtcggaggg gtgctggcct gttacagcct gctggtgaca 60gtcgctttca tcatcttctg
ggtg 8414884DNAartificial sequenceCD28 transmembrane domain (O/SSE)
148atgttctggg tgctcgtggt cgttggcgga gtgctggcct gttacagcct
gctggttacc 60gtggccttca tcatcttttg ggtc 8414928PRTartificial
sequenceCD28 transmembrane domain 149Met Phe Trp Val Leu Val Val
Val Gly Gly Val Leu Ala Cys Tyr Ser1 5 10 15Leu Leu Val Thr Val Ala
Phe Ile Ile Phe Trp Val 20 25150336DNAartificial sequenceCD3-zeta
derived intracellular signaling domain (O/SSE) 150agagtgaagt
tcagcagatc cgccgacgct ccagcctatc agcagggcca aaaccagctg 60tacaacgagc
tgaacctggg gagaagagaa gagtacgacg tgctggataa gcggagaggc
120agagatcctg aaatgggcgg caagcccaga cggaagaatc ctcaagaggg
cctgtataat 180gagctgcaga aagacaagat ggccgaggcc tacagcgaga
tcggaatgaa gggcgagcgc 240agaagaggca agggacacga tggactgtac
cagggcctga gcaccgccac caaggatacc 300tatgacgcac tgcacatgca
ggccctgcca cctaga 3361511074DNAartificial sequencetruncated EGFR
(tEGFR) sequence 151atgcttctcc tggtgacaag ccttctgctc tgtgagttac
cacacccagc attcctcctg 60atcccacgca aagtgtgtaa cggaataggt attggtgaat
ttaaagactc actctccata 120aatgctacga atattaaaca cttcaaaaac
tgcacctcca tcagtggcga tctccacatc 180ctgccggtgg catttagggg
tgactccttc acacatactc ctcctctgga tccacaggaa 240ctggatattc
tgaaaaccgt aaaggaaatc acagggtttt tgctgattca ggcttggcct
300gaaaacagga cggacctcca tgcctttgag aacctagaaa tcatacgcgg
caggaccaag 360caacatggtc agttttctct tgcagtcgtc agcctgaaca
taacatcctt gggattacgc 420tccctcaagg agataagtga tggagatgtg
ataatttcag gaaacaaaaa tttgtgctat 480gcaaatacaa taaactggaa
aaaactgttt gggacctccg gtcagaaaac caaaattata 540agcaacagag
gtgaaaacag ctgcaaggcc acaggccagg tctgccatgc cttgtgctcc
600cccgagggct gctggggccc ggagcccagg gactgcgtct cttgccggaa
tgtcagccga 660ggcagggaat gcgtggacaa gtgcaacctt ctggagggtg
agccaaggga gtttgtggag 720aactctgagt gcatacagtg ccacccagag
tgcctgcctc aggccatgaa catcacctgc 780acaggacggg gaccagacaa
ctgtatccag tgtgcccact acattgacgg cccccactgc 840gtcaagacct
gcccggcagg agtcatggga gaaaacaaca ccctggtctg gaagtacgca
900gacgccggcc atgtgtgcca cctgtgccat ccaaactgca cctacggatg
cactgggcca 960ggtcttgaag gctgtccaac gaatgggcct aagatcccgt
ccatcgccac tgggatggtg 1020ggggccctcc tcttgctgct ggtggtggcc
ctggggatcg gcctcttcat gtga 10741521074DNAartificial
sequencetruncated EGFR (tEGFR) sequence (O/SSE) 152atgctgctcc
tcgtgacaag cctgctcctg tgtgaactcc ctcatccagc ttttctgctc 60attcctcgga
aagtgtgcaa cggcatcggc atcggagagt tcaaggacag cctgagcatc
120aatgccacca acatcaagca cttcaagaat tgcaccagca tcagcggcga
cctgcacatt 180ctgcctgtgg cctttagagg cgacagcttc acccacacac
ctccactgga tccccaagag 240ctggatatcc tgaaaaccgt gaaagagatt
accggattcc tcctgatcca agcctggcca 300gagaacagaa ccgatctgca
cgccttcgag aacctcgaga tcatcagagg ccggaccaaa 360cagcacggcc
agtttagcct ggctgtggtg tctctgaaca tcaccagtct gggcctgaga
420agcctgaaag aaatctccga cggcgacgtg atcatctccg gaaacaagaa
cctgtgctac 480gccaacacca tcaactggaa gaagctgttc ggcacctccg
gccagaaaac aaagatcatc 540tctaaccggg gcgagaacag ctgcaaggcc
accggacaag tttgtcacgc cctgtgtagc 600cctgaaggct gttggggacc
cgaacctaga gactgtgtgt cctgccggaa tgtgtcccgg 660ggcagagaat
gtgtggataa gtgcaacctg ctggaaggcg agccccgcga gtttgtggaa
720aacagcgagt gcatccagtg tcaccccgag tgtctgcccc aggccatgaa
cattacatgc 780accggcagag gccccgacaa ctgtattcag tgcgcccact
acatcgacgg ccctcactgc 840gtgaaaacat gtccagctgg cgtgatggga
gagaacaaca ccctcgtgtg gaagtatgcc 900gacgccggac atgtgtgcca
cctgtgtcac cctaattgca cctacggctg taccggacct 960ggcctggaag
gatgccctac aaacggccct aagatcccca gcattgccac cggaatggtt
1020ggagccctgc tgcttctgtt ggtggtggcc ctcggaatcg gcctgttcat gtga
1074153357PRTartificial sequencetruncated EGFR (tEGFR) sequence
153Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro1
5 10 15Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile
Gly 20 25 30Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys
His Phe 35 40 45Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu
Pro Val Ala 50 55 60Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu
Asp Pro Gln Glu65 70 75 80Leu Asp Ile Leu Lys Thr Val Lys Glu Ile
Thr Gly Phe Leu Leu Ile 85 90 95Gln Ala Trp Pro Glu Asn Arg Thr Asp
Leu His Ala Phe Glu Asn Leu 100 105 110Glu Ile Ile Arg Gly Arg Thr
Lys Gln His Gly Gln Phe Ser Leu Ala 115 120 125Val Val Ser Leu Asn
Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu 130 135 140Ile Ser Asp
Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr145 150 155
160Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys
165 170 175Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala
Thr Gly 180 185 190Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys
Trp Gly Pro Glu 195 200 205Pro Arg Asp Cys Val Ser Cys Arg Asn Val
Ser Arg Gly Arg Glu Cys 210 215 220Val Asp Lys Cys Asn Leu Leu Glu
Gly Glu Pro Arg Glu Phe Val Glu225 230 235 240Asn Ser Glu Cys Ile
Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met 245 250 255Asn Ile Thr
Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala 260 265 270His
Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val 275 280
285Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His
290 295 300Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr
Gly Pro305 310 315 320Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys
Ile Pro Ser Ile Ala 325 330 335Thr Gly Met Val Gly Ala Leu Leu Leu
Leu Leu Val Val Ala Leu Gly 340 345 350Ile Gly Leu Phe Met
355154126DNAartificial sequence4-1BB intracellular co-signaling
sequence 154aagcggggga gaaagaaact gctgtatatt ttcaaacagc cctttatgag
acctgtgcag 60actacccagg aggaagacgg atgcagctgt aggtttcccg aggaagagga
aggaggctgt 120gagctg 126155126DNAartificial sequence4-1BB
intracellular
co-signaling sequence (O/SSE) 155aagcggggca gaaagaagct gctctacatc
ttcaagcagc ccttcatgcg gcccgtgcag 60accacacaag aggaagatgg ctgctcctgc
agattccccg aggaagaaga aggcggctgc 120gagctg 1261561308DNAartificial
sequenceanti-ROR1 CAR 156caggttcagc tgcaagagtc tggccctggc
ctggtcaagc ctagcgaaac actgagcctg 60acctgtaccg tgtctggcgg cagcatctcc
aactactact ggtcctggat cagacagcct 120gccggcaaag gcctggaatg
gatcggcaga atctacacca gcggcagcac caactacaac 180cccagcctga
agtccagagt gaccatgagc gtggacacca gcaagaacca gttctccctg
240aagctgagca gcctgacagc cgccgatacc gccatctact actgtgcccg
gtactacgat 300atcctgaccg gcttcttcga ctactggggc cagggaacac
tggtcacagt ttctagcgga 360ggcggaggat ctggtggcgg aggaagtggc
ggaggcggtt ctgtgatttg gatgacacag 420agccctagcc tgctgagcgc
cagcacaggc gatagcgtga ccatcagctg cagaatgagc 480caggacatca
gcagctacct ggcttggtat cagcagaagc ctggcaaggc ccctgaactg
540ctgatctatg ccgcttccag tctgcagagc ggcgtgccat ctagattttc
cggcagcggc 600tctggcaccg acttcaccct gacaatcagc tccctgcagt
ccgaggactt cgccacctac 660tattgccagc agtacgacag cttccctcca
acctttggcc agggcaccaa ggtggaattc 720aagcgcgagt ctaaatacgg
accgccttgt cctccttgtc ccatgttctg ggtgctcgtg 780gtcgttggcg
gagtgctggc ctgttacagc ctgctggtta ccgtggcctt catcatcttt
840tgggtcaagc ggggcagaaa gaagctgctc tacatcttca agcagccctt
catgcggccc 900gtgcagacca cacaagagga agatggctgc tcctgcagat
tccccgagga agaagaaggc 960ggctgcgagc tgagagtgaa gttcagcaga
tccgccgacg ctccagccta tcagcagggc 1020caaaaccagc tgtacaacga
gctgaacctg gggagaagag aagagtacga cgtgctggat 1080aagcggagag
gcagagatcc tgaaatgggc ggcaagccca gacggaagaa tcctcaagag
1140ggcctgtata atgagctgca gaaagacaag atggccgagg cctacagcga
gatcggaatg 1200aagggcgagc gcagaagagg caagggacac gatggactgt
accagggcct gagcaccgcc 1260accaaggata cctatgacgc actgcacatg
caggccctgc cacctaga 13081571314DNAartificial sequenceanti-ROR1 CAR
157cagctccagc tgcaagaatc tggacctggc ctggtcaagc ccagcgagac
actgtctctg 60acctgtacag tgtccggcgg cagcatcaat agcaccacaa gctactgggc
ctggatcaga 120cagcctcctg gcaaaggcct ggaatggatc ggcaccatct
tctacagcgg caagacctac 180aacaacccca gcctgaagtc cagagtgacc
atgagcgtgg acaccagcaa gaaccacttc 240agcctgaaag tgaacagcgt
gacagccgcc gataccgccg tgtactactg cgccagattc 300gactacggct
tccacgacgc cttcgacatc tggggccagg gcacaatggt cacagtttct
360agcggaggcg gaggatctgg tggcggagga agtggcggag gcggttctga
gattgtgatg 420acacagagcc ccgccactct gagccttagt cctggcgaaa
gagccacact gagctgcaga 480gccagccaga gcatcaccag cgattacctg
agctggtatc agcagaagcc cggacaggct 540cccagactgc tgatctatgg
cgcctctaca agagccaccg gcattcccgc ccgcttttct 600ggctctggaa
gcggcaccga cttcaccctg accatatcta gcctgcagcc tgaggacttc
660gtggtgtact attgccagca ggactacaac ctgtacacct tcggccaggg
gaccaagctg 720gaaatcaaga gagagtctaa atacggaccg ccttgtcctc
cttgtcccat gttctgggtg 780ctcgtggtcg ttggcggagt gctggcctgt
tacagcctgc tggttaccgt ggccttcatc 840atcttttggg tcaagcgggg
cagaaagaag ctgctctaca tcttcaagca gcccttcatg 900cggcccgtgc
agaccacaca agaggaagat ggctgctcct gcagattccc cgaggaagaa
960gaaggcggct gcgagctgag agtgaagttc agcagatccg ccgacgctcc
agcctatcag 1020cagggccaaa accagctgta caacgagctg aacctgggga
gaagagaaga gtacgacgtg 1080ctggataagc ggagaggcag agatcctgaa
atgggcggca agcccagacg gaagaatcct 1140caagagggcc tgtataatga
gctgcagaaa gacaagatgg ccgaggccta cagcgagatc 1200ggaatgaagg
gcgagcgcag aagaggcaag ggacacgatg gactgtacca gggcctgagc
1260accgccacca aggataccta tgacgcactg cacatgcagg ccctgccacc taga
13141581629DNAartificial sequenceanti-ROR1 CAR 158caggttcagc
tgcaagagtc tggccctggc ctggtcaagc ctagcgaaac actgagcctg 60acctgtaccg
tgtctggcgg cagcatctcc aactactact ggtcctggat cagacagcct
120gccggcaaag gcctggaatg gatcggcaga atctacacca gcggcagcac
caactacaac 180cccagcctga agtccagagt gaccatgagc gtggacacca
gcaagaacca gttctccctg 240aagctgagca gcctgacagc cgccgatacc
gccatctact actgtgcccg gtactacgat 300atcctgaccg gcttcttcga
ctactggggc cagggaacac tggtcacagt ttctagcgga 360ggcggaggat
ctggtggcgg aggaagtggc ggaggcggtt ctgtgatttg gatgacacag
420agccctagcc tgctgagcgc cagcacaggc gatagcgtga ccatcagctg
cagaatgagc 480caggacatca gcagctacct ggcttggtat cagcagaagc
ctggcaaggc ccctgaactg 540ctgatctatg ccgcttccag tctgcagagc
ggcgtgccat ctagattttc cggcagcggc 600tctggcaccg acttcaccct
gacaatcagc tccctgcagt ccgaggactt cgccacctac 660tattgccagc
agtacgacag cttccctcca acctttggcc agggcaccaa ggtggaattc
720aagcgcgagt ctaaatacgg accgccttgt cctccttgtc ccggccagcc
aagagagccc 780caggtttaca cactgcctcc aagccaagag gaaatgacca
agaatcaggt gtccctgaca 840tgcctggtca agggcttcta cccctccgat
atcgccgtgg aatgggagag caatggccag 900cctgagaaca actacaagac
cacacctcct gtgctggaca gcgacggcag tttcttcctg 960tatagtagac
tcaccgtgga taaatcaaga tggcaagagg gcaacgtgtt cagctgcagc
1020gtgatgcacg aggccctgca caaccactac acccagaaaa gcctgagcct
gtctctgggc 1080aagatgttct gggtgctcgt ggtcgttggc ggagtgctgg
cctgttacag cctgctggtt 1140accgtggcct tcatcatctt ttgggtcaag
cggggcagaa agaagctgct ctacatcttc 1200aagcagccct tcatgcggcc
cgtgcagacc acacaagagg aagatggctg ctcctgcaga 1260ttccccgagg
aagaagaagg cggctgcgag ctgagagtga agttcagcag atccgccgac
1320gctccagcct atcagcaggg ccaaaaccag ctgtacaacg agctgaacct
ggggagaaga 1380gaagagtacg acgtgctgga taagcggaga ggcagagatc
ctgaaatggg cggcaagccc 1440agacggaaga atcctcaaga gggcctgtat
aatgagctgc agaaagacaa gatggccgag 1500gcctacagcg agatcggaat
gaagggcgag cgcagaagag gcaagggaca cgatggactg 1560taccagggcc
tgagcaccgc caccaaggat acctatgacg cactgcacat gcaggccctg
1620ccacctaga 16291591656DNAartificial sequenceanti-ROR1 CAR
159caggtgcagc tggttcaatc tggcgccgaa gtgaagaaac caggcgcctc
tgtgaaggtg 60tcctgcaagg ccagcggcta cacctttacc agctacggca tcagctgggt
ccgacaggct 120cctggacaag gcttggaatg gatgggctgg atcagcgcct
acaacggcaa caccaaatac 180gcccagaaac tgcagggcag agtgaccatg
accaccgaca ccagcacaag caccgcctac 240atggaactgc ggagcctgag
atccgatgac accgccgtgt actactgcgc cagagatgag 300gacatcctga
ccggctacaa ctactacggc atggacgtgt ggggccaggg cacaacagtg
360acagtttctt ctggcggcgg aggatctggc ggaggtggaa gcggaggcgg
tggatctcaa 420ctggtgctga cacagtctcc tagcgcctct gcttctctgg
gagccagcgt gaagctgacc 480tgtacactgt ctagcggcca cagcagctac
gccattgctt ggcatcagca gcagcccgag 540aagggcccta gatacctgat
gaagctgaac agcgacggca gccactctaa aggcgacggc 600atccccgata
gattcagcgg cagttctagc ggagccgagc gctacctgac aatcagctct
660ctgcaatccg aggacgaggc cgactactac tgtcagacat ggggcaccgg
catcagagtg 720tttggcggag gcaccaagct gacagtgctt ggagagtcta
aatacggacc gccttgtcct 780ccttgtcccg gccagccaag agagccccag
gtttacacac tgcctccaag ccaagaggaa 840atgaccaaga atcaggtgtc
cctgacatgc ctggtcaagg gcttctaccc ctccgatatc 900gccgtggaat
gggagagcaa tggccagcct gagaacaact acaagaccac acctcctgtg
960ctggacagcg acggcagttt cttcctgtat agtagactca ccgtggataa
atcaagatgg 1020caagagggca acgtgttcag ctgcagcgtg atgcacgagg
ccctgcacaa ccactacacc 1080cagaaaagcc tgagcctgtc tctgggcaag
atgttctggg tgctcgtggt cgttggcgga 1140gtgctggcct gttacagcct
gctggttacc gtggccttca tcatcttttg ggtcaagcgg 1200ggcagaaaga
agctgctcta catcttcaag cagcccttca tgcggcccgt gcagaccaca
1260caagaggaag atggctgctc ctgcagattc cccgaggaag aagaaggcgg
ctgcgagctg 1320agagtgaagt tcagcagatc cgccgacgct ccagcctatc
agcagggcca aaaccagctg 1380tacaacgagc tgaacctggg gagaagagaa
gagtacgacg tgctggataa gcggagaggc 1440agagatcctg aaatgggcgg
caagcccaga cggaagaatc ctcaagaggg cctgtataat 1500gagctgcaga
aagacaagat ggccgaggcc tacagcgaga tcggaatgaa gggcgagcgc
1560agaagaggca agggacacga tggactgtac cagggcctga gcaccgccac
caaggatacc 1620tatgacgcac tgcacatgca ggccctgcca cctaga
16561601635DNAartificial sequenceanti-ROR1 CAR 160cagctccagc
tgcaagaatc tggacctggc ctggtcaagc ccagcgagac actgtctctg 60acctgtacag
tgtccggcgg cagcatcaat agcaccacaa gctactgggc ctggatcaga
120cagcctcctg gcaaaggcct ggaatggatc ggcaccatct tctacagcgg
caagacctac 180aacaacccca gcctgaagtc cagagtgacc atgagcgtgg
acaccagcaa gaaccacttc 240agcctgaaag tgaacagcgt gacagccgcc
gataccgccg tgtactactg cgccagattc 300gactacggct tccacgacgc
cttcgacatc tggggccagg gcacaatggt cacagtttct 360agcggaggcg
gaggatctgg tggcggagga agtggcggag gcggttctga gattgtgatg
420acacagagcc ccgccactct gagccttagt cctggcgaaa gagccacact
gagctgcaga 480gccagccaga gcatcaccag cgattacctg agctggtatc
agcagaagcc cggacaggct 540cccagactgc tgatctatgg cgcctctaca
agagccaccg gcattcccgc ccgcttttct 600ggctctggaa gcggcaccga
cttcaccctg accatatcta gcctgcagcc tgaggacttc 660gtggtgtact
attgccagca ggactacaac ctgtacacct tcggccaggg gaccaagctg
720gaaatcaaga gagagtctaa atacggaccg ccttgtcctc cttgtcccgg
ccagccaaga 780gagccccagg tttacacact gcctccaagc caagaggaaa
tgaccaagaa tcaggtgtcc 840ctgacatgcc tggtcaaggg cttctacccc
tccgatatcg ccgtggaatg ggagagcaat 900ggccagcctg agaacaacta
caagaccaca cctcctgtgc tggacagcga cggcagtttc 960ttcctgtata
gtagactcac cgtggataaa tcaagatggc aagagggcaa cgtgttcagc
1020tgcagcgtga tgcacgaggc cctgcacaac cactacaccc agaaaagcct
gagcctgtct 1080ctgggcaaga tgttctgggt gctcgtggtc gttggcggag
tgctggcctg ttacagcctg 1140ctggttaccg tggccttcat catcttttgg
gtcaagcggg gcagaaagaa gctgctctac 1200atcttcaagc agcccttcat
gcggcccgtg cagaccacac aagaggaaga tggctgctcc 1260tgcagattcc
ccgaggaaga agaaggcggc tgcgagctga gagtgaagtt cagcagatcc
1320gccgacgctc cagcctatca gcagggccaa aaccagctgt acaacgagct
gaacctgggg 1380agaagagaag agtacgacgt gctggataag cggagaggca
gagatcctga aatgggcggc 1440aagcccagac ggaagaatcc tcaagagggc
ctgtataatg agctgcagaa agacaagatg 1500gccgaggcct acagcgagat
cggaatgaag ggcgagcgca gaagaggcaa gggacacgat 1560ggactgtacc
agggcctgag caccgccacc aaggatacct atgacgcact gcacatgcag
1620gccctgccac ctaga 16351611656DNAanti-ROR1 CAR 161caggttcagc
tgcttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60tcctgcaagg
ccagcggcta cacctttacc agctacggca tcagctgggt ccgacaggct
120cctggacaag gcttggaatg gatgggctgg atcagcgcct acaccggcaa
taccagatac 180gcccagaaac tgcagggcag agtgaccatg accaccgaca
ccagcacaag caccgcctac 240atggaactgc ggagcctgag atccgatgac
accgccgtgt actactgcgc cagagaagaa 300ggcgccacca ccgactacga
ctactacggc atggatgtgt ggggccaggg aacagccgtg 360acagtttctt
ctggtggcgg aggatctggc ggaggtggaa gcggcggagg cggatctcaa
420ctggttctga cacagagccc aagcgcctct gcatctctgg gagcttccgt
gaagctgacc 480tgcacactgt ctagcggcca cagcagctat gccattgcct
ggcatcagca acagcccgag 540aagggcccta gatacctgat gaagctgaac
agcgacggca gccactctaa aggcgacggc 600atccccgata gattcagcgg
cagttctagc ggagccgagc gctacctgac aatcagctct 660ctgcaatccg
aggacgaggc cgattactac tgtcagacat ggggcaccgg catcagagtg
720tttggcggcg gaacaaagct gaccgtgctg ggcgagtcta aatacggacc
gccttgtcct 780ccttgtcccg gccagccaag agagccccag gtttacacac
tgcctccaag ccaagaggaa 840atgaccaaga atcaggtgtc cctgacatgc
ctggtcaagg gcttctaccc ctccgatatc 900gccgtggaat gggagagcaa
tggccagcct gagaacaact acaagaccac acctcctgtg 960ctggacagcg
acggcagttt cttcctgtat agtagactca ccgtggataa atcaagatgg
1020caagagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa
ccactacacc 1080cagaaaagcc tgagcctgtc tctgggcaag atgttctggg
tgctcgtggt cgttggcgga 1140gtgctggcct gttacagcct gctggttacc
gtggccttca tcatcttttg ggtcaagcgg 1200ggcagaaaga agctgctcta
catcttcaag cagcccttca tgcggcccgt gcagaccaca 1260caagaggaag
atggctgctc ctgcagattc cccgaggaag aagaaggcgg ctgcgagctg
1320agagtgaagt tcagcagatc cgccgacgct ccagcctatc agcagggcca
aaaccagctg 1380tacaacgagc tgaacctggg gagaagagaa gagtacgacg
tgctggataa gcggagaggc 1440agagatcctg aaatgggcgg caagcccaga
cggaagaatc ctcaagaggg cctgtataat 1500gagctgcaga aagacaagat
ggccgaggcc tacagcgaga tcggaatgaa gggcgagcgc 1560agaagaggca
agggacacga tggactgtac cagggcctga gcaccgccac caaggatacc
1620tatgacgcac tgcacatgca ggccctgcca cctaga 165616221PRTartificial
sequencelinker 162Ser Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly1 5 10 15Ser Leu Glu Met Ala 2016322PRTartificial
sequencelinker 163Gly Ser Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly1 5 10 15Gly Ser Leu Glu Met Ala
20164544DNAartificial sequenceEF1alpha promoter with HTLV1 ehancer
164ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc
acagtccccg 60agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg
cgcggggtaa 120actgggaaag tgatgtcgtg tactggctcc gcctttttcc
cgagggtggg ggagaaccgt 180atataagtgc agtagtcgcc gtgaacgttc
tttttcgcaa cgggtttgcc gccagaacac 240agctgaagct tcgaggggct
cgcatctctc cttcacgcgc ccgccgccct acctgaggcc 300gccatccacg
ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg
360cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt
ccggcgctcc 420cttggagcct acctagactc agccggctct ccacgctttg
cctgaccctg cttgctcaac 480tctacgtctt tgtttcgttt tctgttctgc
gccgttacag atccaagctg tgaccggcgc 540ctac 544165588DNAartificial
sequenceWoodchuck Hepatitis Virus (WHP) Posttranscriptional
Regulatory Element (WPRE) 165atcaacctct ggattacaaa atttgtgaaa
gattgactgg tattcttaac tatgttgctc 60cttttacgct atgtggatac gctgctttaa
tgcctttgta tcatgctatt gcttcccgta 120tggctttcat tttctcctcc
ttgtataaat cctggttgct gtctctttat gaggagttgt 180ggcccgttgt
caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
240gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
cccctcccta 300ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
ctggacaggg gctcggctgt 360tgggcactga caattccgtg gtgttgtcgg
ggaaatcatc gtcctttcct tggctgctcg 420cctgtgttgc cacctggatt
ctgcgcggga cgtccttctg ctacgtccct tcggccctca 480atccagcgga
ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
540gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgc
58816621PRTartificial sequenceT2A peptide 166Gly Ser Gly Glu Gly
Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu1 5 10 15Glu Asn Pro Gly
Pro 2016723PRTartificial sequenceE2A peptide 167Gly Ser Gly Gln Cys
Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp1 5 10 15Val Glu Ser Asn
Pro Gly Pro 2016825PRTartificial sequenceF2A peptide 168Gly Ser Gly
Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala1 5 10 15Gly Asp
Val Glu Ser Asn Pro Gly Pro 20 2516972DNAartificial sequenceT2A
peptide 169ctcgagggcg gcggagaggg cagaggaagt cttctaacat gcggtgacgt
ggaggagaat 60cccggcccta gg 721703358DNAhomo
sapiensmisc_featurehuman ROR1 GenBank M97675.1 170gagctggagc
agccgccacc gccgccgccg agggagcccc gggacggcag cccctgggcg 60cagggtgcgc
tgttctcgga gtccgaccca gggcgactca cgcccactgg tgcgacccgg
120acagcctggg actgacccgc cggcccaggc gaggctgcag ccagagggct
gggaagggat 180cgcgctcgcg gcatccagag gcggccaggc ggaggcgagg
gagcaggtta gagggacaaa 240gagctttgca gacgtccccg gcgtcctgcg
agcgccagcg gccgggacga ggcggccggg 300agcccgggaa gagcccgtgg
atgttctgcg cgcggcctgg gagccgccgc cgccgccgcc 360tcagcgagag
gaggaatgca ccggccgcgc cgccgcggga cgcgcccgcc gctcctggcg
420ctgctggccg cgctgctgct ggccgcacgc ggggctgctg cccaagaaac
agagctgtca 480gtcagtgctg aattagtgcc tacctcatca tggaacatct
caagtgaact caacaaagat 540tcttacctga cccttgatga accaatgaat
aacatcacca cgtctctggg ccagacagca 600gaactgcact gcaaagtctc
tgggaatcca cctcccacca tccgctggtt caaaaatgat 660gctcctgtgg
tccaggagcc ccggaggctc tcctttcggt ccaccatcta tggctctcgg
720ctgcggatta gaaacctcga caccacagac acaggctact tccagtgcgt
ggcaacaaac 780ggcaaggagg tggtttcttc cactggagtc ttgtttgtca
agtttggccc ccctcccact 840gcaagtccag gatactcaga tgagtatgaa
gaagatggat tctgtcagcc atacagaggg 900attgcatgtg caagatttat
tggcaaccgc accgtctata tggagtcttt gcacatgcaa 960ggggaaatag
aaaatcagat cacagctgcc ttcactatga ttggcacttc cagtcactta
1020tctgataagt gttctcagtt cgccattcct tccctgtgcc actatgcctt
cccgtactgc 1080gatgaaactt catccgtccc aaagccccgt gacttgtgtc
gcgatgaatg tgaaatcctg 1140gagaatgtcc tgtgtcaaac agagtacatt
tttgcaagat caaatcccat gattctgatg 1200aggctgaaac tgccaaactg
tgaagatctc ccccagccag agagcccaga agctgcgaac 1260tgtatccgga
ttggaattcc catggcagat cctataaata aaaatcacaa gtgttataac
1320agcacaggtg tggactaccg ggggaccgtc agtgtgacca aatcagggcg
ccagtgccag 1380ccatggaatt cccagtatcc ccacacacac actttcaccg
cccttcgttt cccagagctg 1440aatggaggcc attcctactg ccgcaaccca
gggaatcaaa aggaagctcc ctggtgcttc 1500accttggatg aaaactttaa
gtctgatctg tgtgacatcc cagcttgcga ttcaaaggat 1560tccaaggaga
agaataaaat ggaaatcctg tacatactag tgccaagtgt ggccattccc
1620ctggccattg ctttactctt cttcttcatt tgcgtctgtc ggaataacca
gaagtcatcg 1680tcggcaccag tccagaggca accaaaacac gtcagaggtc
aaaatgtgga gatgtcaatg 1740ctgaatgcat ataaacccaa gagcaaggct
aaagagctac ctctttctgc tgtacgcttt 1800atggaagaat tgggtgagtg
tgcctttgga aaaatctata aaggccatct ctatctccca 1860ggcatggacc
atgctcagct ggttgctatc aagaccttga aagactataa caacccccag
1920caatggatgg aatttcaaca agaagcctcc ctaatggcag aactgcacca
ccccaatatt 1980gtctgccttc taggtgccgt cactcaggaa caacctgtgt
gcatgctttt tgagtatatt 2040aatcaggggg atctccatga gttcctcatc
atgagatccc cacactctga tgttggctgc 2100agcagtgatg aagatgggac
tgtgaaatcc agcctggacc acggagattt tctgcacatt 2160gcaattcaga
ttgcagctgg catggaatac ctgtctagtc acttctttgt ccacaaggac
2220cttgcagctc gcaatatttt aatcggagag caacttcatg taaagatttc
agacttgggg 2280ctttccagag aaatttactc cgctgattac tacagggtcc
agagtaagtc cttgctgccc 2340attcgctgga tgccccctga agccatcatg
tatggcaaat tctcttctga ttcagatatc 2400tggtcctttg gggttgtctt
gtgggagatt ttcagttttg gactccagcc atattatgga 2460ttcagtaacc
aggaagtgat tgagatggtg agaaaacggc agctcttacc atgctctgaa
2520gactgcccac ccagaatgta cagcctcatg acagagtgct ggaatgagat
tccttctagg 2580agaccaagat ttaaagatat tcacgtccgg cttcggtcct
gggagggact ctcaagtcac 2640acaagctcta ctactccttc agggggaaat
gccaccacac agacaacctc cctcagtgcc 2700agcccagtga gtaatctcag
taaccccaga tatcctaatt acatgttccc gagccagggt 2760attacaccac
agggccagat tgctggtttc attggcccgc caatacctca gaaccagcga
2820ttcattccca tcaatggata cccaatacct cctggatatg cagcgtttcc
agctgcccac 2880taccagccaa caggtcctcc cagagtgatt cagcactgcc
cacctcccaa gagtcggtcc 2940ccaagcagtg ccagtgggtc gactagcact
ggccatgtga ctagcttgcc ctcatcagga 3000tccaatcagg aagcaaatat
tcctttacta ccacacatgt caattccaaa tcatcctggt 3060ggaatgggta
tcaccgtttt tggcaacaaa tctcaaaaac cctacaaaat tgactcaaag
3120caagcatctt tactaggaga cgccaatatt catggacaca ccgaatctat
gatttctgca 3180gaactgtaaa atgcacaact tttgtaaatg tggtatacag
gacaaactag acggccgtag 3240aaaagattta tattcaaatg tttttattaa
agtaaggttc tcatttagca gacatcgcaa 3300caagtacctt ctgtgaagtt
tcactgtgtc ttaccaagca ggacagacac tcggccag 3358171908PRTmus
musculusMISC_FEATUREMouse ROR1; GenBank No. NP_038873 171Gln Glu
Thr Glu Leu Ser Val Ser Ala Glu Leu Val Pro Thr Ser Ser1 5 10 15Trp
Asn Thr Ser Ser Glu Ile Asp Lys Gly Ser Tyr Leu Thr Leu Asp 20 25
30Glu Pro Met Asn Asn Ile Thr Thr Ser Leu Gly Gln Thr Ala Glu Leu
35 40 45His Cys Lys Val Ser Gly Asn Pro Pro Pro Ser Ile Arg Trp Phe
Lys 50 55 60Asn Asp Ala Pro Val Val Gln Glu Pro Arg Arg Ile Ser Phe
Arg Ala65 70 75 80Thr Asn Tyr Gly Ser Arg Leu Arg Ile Arg Asn Leu
Asp Thr Thr Asp 85 90 95Thr Gly Tyr Phe Gln Cys Val Ala Thr Asn Gly
Lys Lys Val Val Ser 100 105 110Thr Thr Gly Val Leu Phe Val Lys Phe
Gly Pro Pro Pro Thr Ala Ser 115 120 125Pro Gly Ser Ser Asp Glu Tyr
Glu Glu Asp Gly Phe Cys Gln Pro Tyr 130 135 140Arg Gly Ile Ala Cys
Ala Arg Phe Ile Gly Asn Arg Thr Val Tyr Met145 150 155 160Glu Ser
Leu His Met Gln Gly Glu Ile Glu Asn Gln Ile Thr Ala Ala 165 170
175Phe Thr Met Ile Gly Thr Ser Ser His Leu Ser Asp Lys Cys Ser Gln
180 185 190Phe Ala Ile Pro Ser Leu Cys His Tyr Ala Phe Pro Tyr Cys
Asp Glu 195 200 205Thr Ser Ser Val Pro Lys Pro Arg Asp Leu Cys Arg
Asp Glu Cys Glu 210 215 220Val Leu Glu Asn Val Leu Cys Gln Thr Glu
Tyr Ile Phe Ala Arg Ser225 230 235 240Asn Pro Met Ile Leu Met Arg
Leu Lys Leu Pro Asn Cys Glu Asp Leu 245 250 255Pro Gln Pro Glu Ser
Pro Glu Ala Ala Asn Cys Ile Arg Ile Gly Ile 260 265 270Pro Met Ala
Asp Pro Ile Asn Lys Asn His Lys Cys Tyr Asn Ser Thr 275 280 285Gly
Val Asp Tyr Arg Gly Thr Val Ser Val Thr Lys Ser Gly Arg Gln 290 295
300Cys Gln Pro Trp Asn Ser Gln Tyr Pro His Thr His Ser Phe Thr
Ala305 310 315 320Leu Arg Phe Pro Glu Leu Asn Gly Gly His Ser Tyr
Cys Arg Asn Pro 325 330 335Gly Asn Gln Lys Glu Ala Pro Trp Cys Phe
Thr Leu Asp Glu Asn Phe 340 345 350Lys Ser Asp Leu Cys Asp Ile Pro
Ala Cys Asp Ser Lys Asp Ser Lys 355 360 365Glu Lys Asn Lys Met Glu
Ile Leu Tyr Ile Leu Val Pro Ser Val Ala 370 375 380Ile Pro Leu Ala
Ile Ala Phe Leu Phe Phe Phe Ile Cys Val Cys Arg385 390 395 400Asn
Asn Gln Lys Ser Ser Ser Pro Pro Val Gln Arg Gln Pro Lys Pro 405 410
415Val Arg Gly Gln Asn Val Glu Met Ser Met Leu Asn Ala Tyr Lys Pro
420 425 430Lys Ser Lys Ala Lys Glu Leu Pro Leu Ser Ala Val Arg Phe
Met Glu 435 440 445Glu Leu Gly Glu Cys Thr Phe Gly Lys Ile Tyr Lys
Gly His Leu Tyr 450 455 460Leu Pro Gly Met Asp His Ala Gln Leu Val
Ala Ile Lys Thr Leu Lys465 470 475 480Asp Tyr Asn Asn Pro Gln Gln
Trp Thr Glu Phe Gln Gln Glu Ala Ser 485 490 495Leu Met Ala Glu Leu
His His Pro Asn Ile Val Cys Leu Leu Gly Ala 500 505 510Val Thr Gln
Glu Gln Pro Val Cys Met Leu Phe Glu Tyr Met Asn Gln 515 520 525Gly
Asp Leu His Glu Phe Leu Ile Met Arg Ser Pro His Ser Asp Val 530 535
540Gly Cys Ser Ser Asp Glu Asp Gly Thr Val Lys Ser Ser Leu Asp
His545 550 555 560Gly Asp Phe Leu His Ile Ala Ile Gln Ile Ala Ala
Gly Met Glu Tyr 565 570 575Leu Ser Ser His Phe Phe Val His Lys Asp
Leu Ala Ala Arg Asn Ile 580 585 590Leu Ile Gly Glu Gln Leu His Val
Lys Ile Ser Asp Leu Gly Leu Ser 595 600 605Arg Glu Ile Tyr Ser Ala
Asp Tyr Tyr Arg Val Gln Ser Lys Ser Ser 610 615 620Leu Pro Ile Arg
Trp Met Pro Pro Glu Ala Ile Met Tyr Gly Lys Phe625 630 635 640Ser
Ser Asp Ser Asp Ile Trp Ser Phe Gly Val Val Leu Trp Glu Ile 645 650
655Phe Ser Phe Gly Leu Gln Pro Tyr Tyr Gly Phe Ser Asn Gln Glu Val
660 665 670Ile Glu Met Val Arg Lys Arg Gln Leu Leu Pro Cys Ser Glu
Asp Cys 675 680 685Pro Pro Arg Met Tyr Ser Leu Met Thr Glu Cys Trp
Asn Glu Ile Pro 690 695 700Ser Arg Arg Pro Arg Phe Lys Asp Ile His
Val Arg Leu Arg Ser Trp705 710 715 720Glu Gly Leu Ser Ser His Thr
Ser Ser Thr Thr Pro Ser Gly Gly Asn 725 730 735Ala Thr Thr Gln Thr
Thr Ser Leu Ser Ala Ser Pro Val Ser Asn Leu 740 745 750Ser Asn Pro
Arg Phe Pro Asn Tyr Met Phe Pro Ser Gln Gly Ile Thr 755 760 765Pro
Gln Gly Gln Ile Ala Gly Phe Ile Gly Pro Ala Ile Pro Gln Asn 770 775
780Gln Arg Phe Ile Pro Ile Asn Gly Tyr Pro Ile Pro Pro Gly Tyr
Ala785 790 795 800Ala Phe Pro Ala Ala His Tyr Gln Pro Ala Gly Pro
Pro Arg Val Ile 805 810 815Gln His Cys Pro Pro Pro Lys Ser Arg Ser
Pro Ser Ser Ala Ser Gly 820 825 830Ser Thr Ser Thr Gly His Val Ala
Ser Leu Pro Ser Ser Gly Ser Asn 835 840 845Gln Glu Ala Asn Val Pro
Leu Leu Pro His Met Ser Ile Pro Asn His 850 855 860Pro Gly Gly Met
Gly Ile Thr Val Phe Gly Asn Lys Ser Gln Lys Pro865 870 875 880Tyr
Lys Ile Asp Ser Lys Gln Ser Ser Leu Leu Gly Asp Ser His Ile 885 890
895His Gly His Thr Glu Ser Met Ile Ser Ala Glu Val 900
905172118PRTartificial sequence2A2 VH 172Gln Val Gln Leu Gln Gln
Ser Gly Ala Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Thr Leu Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr 20 25 30Glu Met His Trp
Val Ile Gln Thr Pro Val His Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile
Asp Pro Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys Gly
Lys Ala Ile Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Thr Gly Tyr Tyr Asp Tyr Asp Ser Phe Thr Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ala 115173107PRTartificial
sequence2A2 VL 173Asp Ile Val Met Thr Gln Ser Gln Lys Ile Met Ser
Thr Thr Val Gly1 5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln
Asn Val Asp Ala Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Asn Arg Tyr Thr Gly
Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Asn Met Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr
Phe Cys Gln Gln Tyr Asp Ile Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 100 105174116PRTartificial sequence99961
humanized VH 174Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ala
Phe Thr Ala Tyr 20 25 30Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45Gly Ser Phe Asp Pro Tyr Asp Gly Gly Ser
Ser Tyr Asn Gln Lys Phe 50 55 60Lys Asp Arg Leu Thr Ile Ser Lys Asp
Thr Ser Lys Asn Gln Val Val65 70 75 80Leu Thr Met Thr Asn Met Asp
Pro Val Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Ala Arg Gly Trp Tyr Tyr
Phe Asp Tyr Trp Gly His Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115175116PRTartificial sequence99961 humanized VH 175Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Tyr Ala Phe Thr Ala Tyr 20 25 30Asn
Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Ser Phe Asp Pro Tyr Asp Gly Gly Ser Ser Tyr Asn Gln Lys Phe
50 55 60Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val
Val65 70 75 80Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr
Tyr Tyr Cys 85 90 95Ala Arg Gly Trp Tyr Tyr Phe Asp Tyr Trp Gly His
Gly Thr Leu Val 100 105 110Thr Val Ser Ser 115176116PRTartificial
sequence99961 humanized VH 176Gln Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Tyr Ala Phe Thr Ala Tyr 20 25 30Asn Ile His Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Ser Phe Asp Pro Tyr
Asp Gly Gly Ser Ser Tyr Asn Gln Lys Phe 50 55 60Lys Asp Arg Leu Thr
Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val65 70 75 80Leu Thr Met
Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Ala Arg
Gly Trp Tyr Tyr Phe Asp Tyr Trp Gly His Gly Thr Leu Val 100 105
110Thr Val Ser Ser 115177116PRTartificial sequence99961 humanized
VH 177Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ala Phe Thr
Ala Tyr 20 25 30Asn Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45Gly Ser Phe Asp Pro Tyr Asp Gly Gly Ser Ser Tyr
Asn Gln Lys Phe 50 55 60Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser
Lys Asn Gln Val Val65 70 75 80Leu Thr Met Thr Asn Met Asp Pro Val
Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Ala Arg Gly Trp Tyr Tyr Phe Asp
Tyr Trp Gly His Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115178107PRTartificial sequence99961 humanized VL 178Asp Ile Val
Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro
Ala Ser Ile Ser Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Pro Arg Phe Ser Gly
50 55 60Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Ile Glu
Ser65 70 75 80Glu Asp Ala Ala Tyr Tyr Phe Cys Gln Gln His Asp Glu
Ser Pro Tyr 85 90 95Thr Phe Gly Glu Gly Thr Lys Val Glu Ile Lys 100
105179107PRTartificial sequence99961 humanized VL 179Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro
Ala Ser Ile Ser Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Pro Arg Phe Ser Gly
50 55 60Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Ile Glu
Ser65 70 75 80Glu Asp Ala Ala Tyr Tyr Phe Cys Gln Gln His Asp Glu
Ser Pro Tyr 85 90 95Thr Phe Gly Glu Gly Thr Lys Val Glu Ile Lys 100
105180107PRTartificial sequence99961 humanized VL 180Asp Ile Val
Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro
Ala Ser Ile Ser Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Pro Arg Phe Ser Gly
50 55 60Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Ile Glu
Ser65 70 75 80Glu Asp Ala Ala Tyr Tyr Phe Cys Gln Gln His Asp Glu
Ser Pro Tyr 85 90 95Thr Phe Gly Glu Gly Thr Lys Val Glu Ile Lys 100
105181107PRTartificial sequence99961 humanized VL 181Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro
Ala Ser Ile Ser Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Pro Arg Phe Ser Gly
50 55 60Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Ile Glu
Ser65 70 75 80Glu Asp Ala Ala Tyr Tyr Phe Cys Gln Gln His Asp Glu
Ser Pro Tyr 85 90 95Thr Phe Gly Glu Gly Thr Lys Val Glu Ile Lys 100
105182336DNAartificial sequenceCD3-zeta derived intracellular
signaling domain 182agagtcaagt tttccaggtc cgccgacgct ccagcctacc
agcaggggca gaaccagctg 60tacaacgagc tgaacctggg cagaagggaa gagtacgacg
tcctggataa gcggagaggc 120cgggaccctg agatgggcgg caagcctcgg
cggaagaacc cccaggaagg cctgtataac 180gaactgcaga aagacaagat
ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 240aggcggggca
agggccacga cggcctgtat cagggcctgt ccaccgccac caaggatacc
300tacgacgccc tgcacatgca ggccctgccc ccaagg 336183123DNAartificial
sequenceCD28 endodomain 183aggagtaaga ggagcaggct cctgcacagt
gactacatga acatgactcc ccgccgcccc 60gggcccaccc gcaagcatta ccagccctat
gccccaccac gcgacttcgc agcctatcgc 120tcc 123184436PRTartificial
sequenceanti-ROR1 CAR 184Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Gly Ser Ile Ser Asn Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro
Ala Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Tyr Thr Ser Gly
Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Met Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser
Leu Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95Arg Tyr Tyr
Asp Ile Leu Thr Gly Phe Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120
125Ser Gly Gly Gly Gly Ser Val Ile Trp Met Thr Gln Ser Pro Ser Leu
130 135 140Leu Ser Ala Ser Thr Gly Asp Ser Val Thr Ile Ser Cys Arg
Met Ser145 150 155 160Gln Asp Ile Ser Ser Tyr Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys 165 170 175Ala Pro Glu Leu Leu Ile Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val 180 185 190Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205Ile Ser Ser
Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215 220Tyr
Asp Ser Phe Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Phe225 230
235 240Lys Arg Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met
Phe 245 250 255Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr
Ser Leu Leu 260 265 270Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys
Arg Gly Arg Lys Lys 275 280 285Leu Leu Tyr Ile Phe Lys Gln Pro Phe
Met Arg Pro Val Gln Thr Thr 290 295 300Gln Glu Glu Asp Gly Cys Ser
Cys Arg Phe Pro Glu Glu Glu Glu Gly305 310 315 320Gly Cys Glu Leu
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 325 330 335Tyr Gln
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 340 345
350Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
355 360 365Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
Tyr Asn 370 375 380Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
Glu Ile Gly Met385 390 395 400Lys Gly Glu Arg Arg Arg Gly Lys Gly
His Asp Gly Leu Tyr Gln Gly 405 410 415Leu Ser Thr Ala Thr Lys Asp
Thr Tyr Asp Ala Leu His Met Gln Ala 420 425 430Leu Pro Pro Arg
435185438PRTartificial sequenceanti-ROR1 CAR 185Gln Leu Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gly Ser Ile Asn Ser Thr 20 25 30Thr Ser Tyr
Trp Ala Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45Trp Ile
Gly Thr Ile Phe Tyr Ser Gly Lys Thr Tyr Asn Asn Pro Ser 50 55 60Leu
Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn His Phe65 70 75
80Ser Leu Lys Val Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95Cys Ala Arg Phe Asp Tyr Gly Phe His Asp Ala Phe Asp Ile Trp
Gly 100 105 110Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly 115 120 125Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val
Met Thr Gln Ser Pro 130 135 140Ala Thr Leu Ser Leu Ser Pro Gly Glu
Arg Ala Thr Leu Ser Cys Arg145 150 155 160Ala Ser Gln Ser Ile Thr
Ser Asp Tyr Leu Ser Trp Tyr Gln Gln Lys 165 170 175Pro Gly Gln Ala
Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala 180 185 190Thr Gly
Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200
205Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Val Val Tyr Tyr
210 215 220Cys Gln Gln Asp Tyr Asn Leu Tyr Thr Phe Gly Gln Gly Thr
Lys Leu225 230 235 240Glu Ile Lys Arg Glu Ser Lys Tyr Gly Pro Pro
Cys Pro Pro Cys Pro 245 250 255Met Phe Trp Val Leu Val Val Val Gly
Gly Val Leu Ala Cys Tyr Ser 260 265 270Leu Leu Val Thr Val Ala Phe
Ile Ile Phe Trp Val Lys Arg Gly Arg 275 280 285Lys Lys Leu Leu Tyr
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 290 295 300Thr Thr Gln
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu305 310 315
320Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
325 330 335Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
Asn Leu 340 345 350Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
Arg Gly Arg Asp 355 360 365Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
Asn Pro Gln Glu Gly Leu 370 375 380Tyr Asn Glu Leu Gln Lys Asp Lys
Met Ala Glu Ala Tyr Ser Glu Ile385 390 395 400Gly Met Lys Gly Glu
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 405 410 415Gln Gly Leu
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 420 425 430Gln
Ala Leu Pro Pro Arg 435186543PRTartificial sequenceanti-ROR1 CAR
186Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn
Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Ala Gly Lys Gly Leu Glu
Trp Ile 35 40 45Gly Arg Ile Tyr Thr Ser Gly Ser Thr Asn Tyr Asn Pro
Ser Leu Lys 50 55 60Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn
Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Leu Thr Ala Ala Asp Thr
Ala Ile Tyr Tyr Cys Ala 85 90 95Arg Tyr Tyr Asp Ile Leu Thr Gly Phe
Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125Ser Gly Gly Gly Gly
Ser Val Ile Trp Met Thr Gln Ser Pro Ser Leu 130 135 140Leu Ser Ala
Ser Thr Gly Asp Ser Val Thr Ile Ser Cys Arg Met Ser145 150 155
160Gln Asp Ile Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
165 170 175Ala Pro Glu Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser
Gly Val 180 185 190Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr 195 200 205Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln 210 215 220Tyr Asp Ser Phe Pro Pro Thr Phe
Gly Gln Gly Thr Lys Val Glu Phe225 230 235 240Lys Arg Glu Ser Lys
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln 245 250 255Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 260 265 270Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 275 280
285Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
290 295 300Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu305 310 315 320Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val 325 330 335Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln 340 345 350Lys Ser Leu Ser Leu Ser Leu
Gly Lys Met Phe Trp Val Leu Val Val 355 360 365Val Gly Gly Val Leu
Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe 370 375 380Ile Ile Phe
Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe385 390 395
400Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
405 410 415Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
Leu Arg 420 425 430Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
Gln Gln Gly Gln 435 440 445Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
Arg Arg Glu Glu Tyr Asp 450 455 460Val Leu Asp Lys Arg Arg Gly Arg
Asp Pro Glu Met Gly Gly Lys Pro465 470 475 480Arg Arg Lys Asn Pro
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 485 490 495Lys Met Ala
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 500 505 510Arg
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 515 520
525Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 530
535 540187552PRTartificial sequenceanti-ROR1 CAR 187Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Gly Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Ser Ala Tyr Asn Gly Asn Thr Lys Tyr Ala Gln Lys Leu 50 55
60Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Asp Glu Asp Ile Leu Thr Gly Tyr Asn Tyr Tyr Gly
Met Asp 100 105 110Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
Gly Gly Gly Gly 115 120 125Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gln Leu Val Leu Thr 130 135 140Gln Ser Pro Ser Ala Ser Ala Ser
Leu Gly Ala Ser Val Lys Leu Thr145 150 155 160Cys Thr Leu Ser Ser
Gly His Ser Ser Tyr Ala Ile Ala Trp His Gln 165 170 175Gln Gln Pro
Glu Lys Gly Pro Arg Tyr Leu Met Lys Leu Asn Ser Asp 180 185 190Gly
Ser His Ser Lys Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser 195 200
205Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu
210 215 220Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Gly Thr Gly Ile
Arg Val225 230 235 240Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
Glu Ser Lys Tyr Gly 245 250 255Pro Pro Cys Pro Pro Cys Pro Gly Gln
Pro Arg Glu Pro Gln Val Tyr 260 265 270Thr Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys Asn Gln Val Ser Leu 275 280 285Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 290 295 300Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val305 310 315
320Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
325 330 335Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His 340 345 350Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu 355 360 365Gly Lys Met Phe Trp Val Leu Val Val Val
Gly Gly Val Leu Ala Cys 370 375 380Tyr Ser Leu Leu Val Thr Val Ala
Phe Ile Ile Phe Trp Val Lys Arg385 390 395 400Gly Arg Lys Lys Leu
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 405 410 415Val Gln Thr
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 420 425 430Glu
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 435 440
445Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
450 455 460Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
Arg Gly465 470 475 480Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
Lys Asn Pro Gln Glu 485 490 495Gly Leu Tyr Asn Glu Leu Gln Lys Asp
Lys Met Ala Glu Ala Tyr Ser 500 505 510Glu Ile Gly Met Lys Gly Glu
Arg Arg Arg Gly Lys Gly His Asp Gly 515 520 525Leu Tyr Gln Gly Leu
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 530 535 540His Met Gln
Ala Leu Pro Pro Arg545 550188545PRTartificial sequenceanti-ROR1 CAR
188Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Asn Ser
Thr 20 25 30Thr Ser Tyr Trp Ala Trp Ile Arg Gln Pro Pro Gly Lys Gly
Leu Glu 35 40 45Trp Ile Gly Thr Ile Phe Tyr Ser Gly Lys Thr Tyr Asn
Asn Pro Ser 50 55 60Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser
Lys Asn His Phe65 70 75 80Ser Leu Lys Val Asn Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg Phe Asp Tyr Gly Phe His
Asp Ala Phe Asp Ile Trp Gly 100 105 110Gln Gly Thr Met Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly Ser Gly Gly
Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140Ala Thr Leu
Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg145 150 155
160Ala Ser Gln Ser Ile Thr Ser Asp Tyr Leu Ser Trp Tyr Gln Gln Lys
165 170 175Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr
Arg Ala 180 185 190Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe 195 200 205Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
Asp Phe Val Val Tyr Tyr 210 215 220Cys Gln Gln Asp Tyr Asn Leu Tyr
Thr Phe Gly Gln Gly Thr Lys Leu225 230 235 240Glu Ile Lys Arg Glu
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 245 250 255Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 260 265 270Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 275 280
285Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
290 295 300Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe305 310 315 320Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
Arg Trp Gln Glu Gly 325 330 335Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr 340 345 350Thr Gln Lys Ser Leu Ser Leu
Ser Leu Gly Lys Met Phe Trp Val Leu 355 360 365Val Val Val Gly Gly
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 370 375 380Ala Phe Ile
Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr385 390 395
400Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
405 410 415Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
Cys Glu 420 425 430Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
Ala Tyr Gln Gln 435 440 445Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
Leu Gly Arg Arg Glu Glu 450 455 460Tyr Asp Val Leu Asp Lys Arg Arg
Gly Arg Asp Pro Glu Met Gly Gly465 470 475 480Lys Pro Arg Arg Lys
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 485 490 495Lys Asp Lys
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 500 505 510Arg
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 515 520
525Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
530 535 540Arg545189552PRTartificial sequenceanti-ROR1 CAR 189Gln
Val Gln Leu Leu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Trp Ile Ser Ala Tyr Thr Gly Asn Thr Arg Tyr Ala Gln
Lys Leu 50 55 60Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Glu Gly Ala Thr Thr Asp Tyr
Asp Tyr Tyr Gly Met Asp 100 105
110Val Trp Gly Gln Gly Thr Ala Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Leu Val
Leu Thr 130 135 140Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala Ser
Val Lys Leu Thr145 150 155 160Cys Thr Leu Ser Ser Gly His Ser Ser
Tyr Ala Ile Ala Trp His Gln 165 170 175Gln Gln Pro Glu Lys Gly Pro
Arg Tyr Leu Met Lys Leu Asn Ser Asp 180 185 190Gly Ser His Ser Lys
Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser 195 200 205Ser Ser Gly
Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu 210 215 220Asp
Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Gly Thr Gly Ile Arg Val225 230
235 240Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu Ser Lys Tyr
Gly 245 250 255Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg Glu Pro
Gln Val Tyr 260 265 270Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu 275 280 285Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 290 295 300Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val305 310 315 320Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 325 330 335Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 340 345
350Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
355 360 365Gly Lys Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu
Ala Cys 370 375 380Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
Trp Val Lys Arg385 390 395 400Gly Arg Lys Lys Leu Leu Tyr Ile Phe
Lys Gln Pro Phe Met Arg Pro 405 410 415Val Gln Thr Thr Gln Glu Glu
Asp Gly Cys Ser Cys Arg Phe Pro Glu 420 425 430Glu Glu Glu Gly Gly
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 435 440 445Asp Ala Pro
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 450 455 460Asn
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly465 470
475 480Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
Glu 485 490 495Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
Ala Tyr Ser 500 505 510Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
Lys Gly His Asp Gly 515 520 525Leu Tyr Gln Gly Leu Ser Thr Ala Thr
Lys Asp Thr Tyr Asp Ala Leu 530 535 540His Met Gln Ala Leu Pro Pro
Arg545 55019048DNAartificial sequenceCD33 signal sequence (O/SSE)
190atgcctctgc tgctgcttct gcctcttctt tgggctggtg ctctggct
4819148DNAartificial sequenceCD33 signal sequence GenBank M23197.1
191atgccgctgc tgctactgct gcccctgctg tgggcagggg ccctggct
4819239DNAartificial sequenceSpacer (IgG4 hinge) 192gaatctaagt
acggaccgcc ctgcccccct tgccctatg 39193357DNAartificial
sequenceHinge-CH3 spacer 193gaatctaagt acggaccgcc ttgtcctcca
tgtcctggcc agccaagaga accccaggtg 60tacacactgc ctccaagcca agaggaaatg
accaagaacc aggtgtccct gacctgcctg 120gtcaagggct tctacccttc
cgatatcgcc gtggaatggg agagcaatgg ccagcctgag 180aacaactaca
agaccacacc tcctgtgctg gacagcgacg gctcattctt cctgtacagc
240cggctgaccg tggacaagag cagatggcaa gagggcaacg tgttcagctg
cagcgtgatg 300cacgaggccc tgcacaacca ctacacccag aagtctctga
gcctgagcct gggcaag 357194229PRTartificial sequenceIgG4/IgG2 hinge-
IgG2/IgG4 CH2- IgG4 CH3 spacer 194Glu Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro Pro Val1 5 10 15Ala Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45Gln Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr65 70 75 80Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 100 105
110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
115 120 125Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
Gln Val 130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro 165 170 175Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr 180 185 190Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 195 200 205Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220Ser
Leu Gly Lys Met225195687DNAartificial sequenceIgG4/IgG2 hinge-
IgG2/IgG4 CH2- IgG4 CH3 spacer 195gaatctaagt acggaccgcc ctgccctccc
tgccctgctc ctcctgtggc tggaccaagc 60gtgttcctgt ttccacctaa gcctaaagat
accctgatga tttcccgcac acctgaagtg 120acttgcgtgg tcgtggacgt
gagccaggag gatccagaag tgcagttcaa ctggtacgtg 180gacggcgtgg
aagtccacaa tgctaagact aaaccccgag aggaacagtt tcagtcaact
240taccgggtcg tgagcgtgct gaccgtcctg catcaggatt ggctgaacgg
gaaggagtat 300aagtgcaaag tgtctaataa gggactgcct agctccatcg
agaaaacaat tagtaaggca 360aaagggcagc ctcgagaacc acaggtgtat
accctgcccc ctagccagga ggaaatgacc 420aagaaccagg tgtccctgac
atgtctggtc aaaggcttct atccaagtga catcgccgtg 480gagtgggaat
caaatgggca gcccgagaac aattacaaga ccacaccacc cgtgctggac
540tctgatggaa gtttctttct gtattccagg ctgaccgtgg ataaatctcg
ctggcaggag 600ggcaacgtgt tctcttgcag tgtcatgcac gaagccctgc
acaatcatta tacacagaag 660tcactgagcc tgtccctggg caaaatg
687196687DNAartificial sequenceIgG4/IgG2 hinge- IgG2/IgG4 CH2- IgG4
CH3 spacer O/SSE 196gagtctaaat acggaccgcc ttgtcctcct tgtcccgctc
ctcctgttgc cggaccttcc 60gtgttcctgt ttcctccaaa gcctaaggac accctgatga
tcagcaggac ccctgaagtg 120acctgcgtgg tggtggatgt gtcccaagag
gatcccgagg tgcagttcaa ctggtatgtg 180gacggcgtgg aagtgcacaa
cgccaagacc aagcctagag aggaacagtt ccagagcacc 240tacagagtgg
tgtccgtgct gacagtgctg caccaggatt ggctgaacgg caaagagtac
300aagtgcaagg tgtccaacaa gggcctgcct agcagcatcg agaaaaccat
ctccaaggcc 360aagggccagc caagagagcc ccaggtttac acactgcctc
caagccaaga ggaaatgacc 420aagaatcagg tgtccctgac atgcctggtc
aagggcttct acccctccga tatcgccgtg 480gaatgggaga gcaatggcca
gcctgagaac aactacaaga ccacacctcc tgtgctggac 540agcgacggca
gtttcttcct gtatagtaga ctcaccgtgg ataaatcaag atggcaagag
600ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgc acaaccacta
cacccagaaa 660agcctgagcc tgtctctggg caagatg 68719781DNAartificial
sequenceCD28 transmembrane domain 197ttttgggtgc tggtcgtggt
cggaggggtg ctggcctgtt acagcctgct ggtgacagtc 60gctttcatca tcttctgggt
g 8119881DNAartificial sequenceCD28 transmembrane domain (O/SSE)
198ttctgggtgc tcgtggtcgt tggcggagtg ctggcctgtt acagcctgct
ggttaccgtg 60gccttcatca tcttttgggt c 8119915PRTartificial
sequenceROR1 epitope 1 199Phe Arg Ser Thr Ile Tyr Gly Ser Arg Leu
Arg Ile Arg Asn Leu1 5 10 1520013PRTartificial sequenceROR1 epitope
2 200Leu Ser Val Ser Ala Glu Leu Val Pro Thr Ser Ser Trp1 5
1020114PRTartificial sequenceROR1 epitope 3 201His Cys Lys Val Ser
Gly Asn Pro Pro Pro Thr Ile Arg Trp1 5 1020226PRTartificial
sequenceROR1 epitope 4 202Trp Phe Lys Asn Asp Ala Pro Val Val Gln
Glu Pro Arg Arg Leu Ser1 5 10 15Phe Arg Ser Thr Ile Tyr Gly Ser Arg
Leu 20 252039PRTartificial sequenceROR1 epitope 5 203Val Ser Ser
Thr Gly Val Leu Phe Val1 520413PRTartificial sequenceROR1 epitope 6
204Leu Phe Val Lys Phe Gly Pro Pro Pro Thr Ala Ser Pro1 5
1020511PRTartificial sequenceROR1 epitope 7 205Asp Glu Tyr Glu Glu
Asp Gly Phe Cys Gln Pro1 5 1020614PRTartificial sequenceROR1
epitope 8 206Gln Pro Tyr Arg Gly Ile Ala Cys Ala Arg Phe Ile Gly
Asn1 5 1020714PRTartificial sequenceROR1 epitope 9 207Ser Gln Phe
Ala Ile Pro Ser Leu Cys His Tyr Ala Phe Pro1 5 1020812PRTartificial
sequenceROR1 epitope 10 208Ala Phe Pro Tyr Cys Asp Glu Thr Ser Ser
Val Pro1 5 1020920PRTartificial sequenceROR1 epitope 11 209Asn Val
Leu Cys Gln Thr Glu Tyr Ile Phe Ala Arg Ser Asn Pro Met1 5 10 15Ile
Leu Met Arg 2021012PRTartificial sequenceROR1 epitope 12 210Leu Lys
Leu Pro Asn Cys Glu Asp Leu Pro Gln Pro1 5 1021115PRTartificial
sequenceROR1 epitope 13 211Pro Glu Ser Pro Glu Ala Ala Asn Cys Ile
Arg Ile Gly Ile Pro1 5 10 1521214PRTartificial sequenceROR1 epitope
14 212Val Asp Tyr Arg Gly Thr Val Ser Val Thr Lys Ser Gly Arg1 5
1021314PRTartificial sequenceROR1 epitope 15 213Ser Gln Tyr Pro His
Thr His Thr Phe Thr Ala Leu Arg Phe1 5 1021415PRTartificial
sequenceROR1 epitope 16 214Asp Ser Lys Glu Lys Asn Lys Met Glu Ile
Leu Tyr Ile Leu Val1 5 10 15215937PRThomo sapiensMISC_FEATUREROR1
(Uniprot Q01973) 215Met His Arg Pro Arg Arg Arg Gly Thr Arg Pro Pro
Leu Leu Ala Leu1 5 10 15Leu Ala Ala Leu Leu Leu Ala Ala Arg Gly Ala
Ala Ala Gln Glu Thr 20 25 30Glu Leu Ser Val Ser Ala Glu Leu Val Pro
Thr Ser Ser Trp Asn Ile 35 40 45Ser Ser Glu Leu Asn Lys Asp Ser Tyr
Leu Thr Leu Asp Glu Pro Met 50 55 60Asn Asn Ile Thr Thr Ser Leu Gly
Gln Thr Ala Glu Leu His Cys Lys65 70 75 80Val Ser Gly Asn Pro Pro
Pro Thr Ile Arg Trp Phe Lys Asn Asp Ala 85 90 95Pro Val Val Gln Glu
Pro Arg Arg Leu Ser Phe Arg Ser Thr Ile Tyr 100 105 110Gly Ser Arg
Leu Arg Ile Arg Asn Leu Asp Thr Thr Asp Thr Gly Tyr 115 120 125Phe
Gln Cys Val Ala Thr Asn Gly Lys Glu Val Val Ser Ser Thr Gly 130 135
140Val Leu Phe Val Lys Phe Gly Pro Pro Pro Thr Ala Ser Pro Gly
Tyr145 150 155 160Ser Asp Glu Tyr Glu Glu Asp Gly Phe Cys Gln Pro
Tyr Arg Gly Ile 165 170 175Ala Cys Ala Arg Phe Ile Gly Asn Arg Thr
Val Tyr Met Glu Ser Leu 180 185 190His Met Gln Gly Glu Ile Glu Asn
Gln Ile Thr Ala Ala Phe Thr Met 195 200 205Ile Gly Thr Ser Ser His
Leu Ser Asp Lys Cys Ser Gln Phe Ala Ile 210 215 220Pro Ser Leu Cys
His Tyr Ala Phe Pro Tyr Cys Asp Glu Thr Ser Ser225 230 235 240Val
Pro Lys Pro Arg Asp Leu Cys Arg Asp Glu Cys Glu Ile Leu Glu 245 250
255Asn Val Leu Cys Gln Thr Glu Tyr Ile Phe Ala Arg Ser Asn Pro Met
260 265 270Ile Leu Met Arg Leu Lys Leu Pro Asn Cys Glu Asp Leu Pro
Gln Pro 275 280 285Glu Ser Pro Glu Ala Ala Asn Cys Ile Arg Ile Gly
Ile Pro Met Ala 290 295 300Asp Pro Ile Asn Lys Asn His Lys Cys Tyr
Asn Ser Thr Gly Val Asp305 310 315 320Tyr Arg Gly Thr Val Ser Val
Thr Lys Ser Gly Arg Gln Cys Gln Pro 325 330 335Trp Asn Ser Gln Tyr
Pro His Thr His Thr Phe Thr Ala Leu Arg Phe 340 345 350Pro Glu Leu
Asn Gly Gly His Ser Tyr Cys Arg Asn Pro Gly Asn Gln 355 360 365Lys
Glu Ala Pro Trp Cys Phe Thr Leu Asp Glu Asn Phe Lys Ser Asp 370 375
380Leu Cys Asp Ile Pro Ala Cys Asp Ser Lys Asp Ser Lys Glu Lys
Asn385 390 395 400Lys Met Glu Ile Leu Tyr Ile Leu Val Pro Ser Val
Ala Ile Pro Leu 405 410 415Ala Ile Ala Leu Leu Phe Phe Phe Ile Cys
Val Cys Arg Asn Asn Gln 420 425 430Lys Ser Ser Ser Ala Pro Val Gln
Arg Gln Pro Lys His Val Arg Gly 435 440 445Gln Asn Val Glu Met Ser
Met Leu Asn Ala Tyr Lys Pro Lys Ser Lys 450 455 460Ala Lys Glu Leu
Pro Leu Ser Ala Val Arg Phe Met Glu Glu Leu Gly465 470 475 480Glu
Cys Ala Phe Gly Lys Ile Tyr Lys Gly His Leu Tyr Leu Pro Gly 485 490
495Met Asp His Ala Gln Leu Val Ala Ile Lys Thr Leu Lys Asp Tyr Asn
500 505 510Asn Pro Gln Gln Trp Thr Glu Phe Gln Gln Glu Ala Ser Leu
Met Ala 515 520 525Glu Leu His His Pro Asn Ile Val Cys Leu Leu Gly
Ala Val Thr Gln 530 535 540Glu Gln Pro Val Cys Met Leu Phe Glu Tyr
Ile Asn Gln Gly Asp Leu545 550 555 560His Glu Phe Leu Ile Met Arg
Ser Pro His Ser Asp Val Gly Cys Ser 565 570 575Ser Asp Glu Asp Gly
Thr Val Lys Ser Ser Leu Asp His Gly Asp Phe 580 585 590Leu His Ile
Ala Ile Gln Ile Ala Ala Gly Met Glu Tyr Leu Ser Ser 595 600 605His
Phe Phe Val His Lys Asp Leu Ala Ala Arg Asn Ile Leu Ile Gly 610 615
620Glu Gln Leu His Val Lys Ile Ser Asp Leu Gly Leu Ser Arg Glu
Ile625 630 635 640Tyr Ser Ala Asp Tyr Tyr Arg Val Gln Ser Lys Ser
Leu Leu Pro Ile 645 650 655Arg Trp Met Pro Pro Glu Ala Ile Met Tyr
Gly Lys Phe Ser Ser Asp 660 665 670Ser Asp Ile Trp Ser Phe Gly Val
Val Leu Trp Glu Ile Phe Ser Phe 675 680 685Gly Leu Gln Pro Tyr Tyr
Gly Phe Ser Asn Gln Glu Val Ile Glu Met 690 695 700Val Arg Lys Arg
Gln Leu Leu Pro Cys Ser Glu Asp Cys Pro Pro Arg705 710 715 720Met
Tyr Ser Leu Met Thr Glu Cys Trp Asn Glu Ile Pro Ser Arg Arg 725 730
735Pro Arg Phe Lys Asp Ile His Val Arg Leu Arg Ser Trp Glu Gly Leu
740 745 750Ser Ser His Thr Ser Ser Thr Thr Pro Ser Gly Gly Asn Ala
Thr Thr 755 760 765Gln Thr Thr Ser Leu Ser Ala Ser Pro Val Ser Asn
Leu Ser Asn Pro 770 775 780Arg Tyr Pro Asn Tyr Met Phe Pro Ser Gln
Gly Ile Thr Pro Gln Gly785 790 795 800Gln Ile Ala Gly Phe Ile Gly
Pro Pro Ile Pro Gln Asn Gln Arg Phe 805 810 815Ile Pro Ile Asn Gly
Tyr Pro Ile Pro Pro Gly Tyr Ala Ala Phe Pro 820 825 830Ala Ala His
Tyr Gln Pro Thr Gly Pro Pro Arg Val Ile Gln His Cys 835 840 845Pro
Pro Pro Lys Ser Arg Ser Pro Ser Ser Ala Ser Gly Ser Thr Ser 850 855
860Thr Gly His Val Thr Ser Leu Pro Ser Ser Gly Ser Asn Gln Glu
Ala865 870 875 880Asn Ile Pro Leu Leu Pro His Met Ser Ile Pro Asn
His Pro Gly Gly 885 890 895Met Gly Ile Thr Val Phe Gly Asn Lys Ser
Gln Lys Pro Tyr Lys Ile 900 905 910Asp Ser Lys Gln Ala Ser Leu Leu
Gly Asp Ala Asn Ile His Gly His 915 920 925Thr Glu Ser Met Ile Ser
Ala Glu Leu 930 935216937PRTMacaca mulattaMISC_FEATUREROR1 (Uniprot
F6RUP2) 216Met His Arg Pro Arg Arg Arg Gly Thr Arg Pro Pro Leu Leu
Ala Leu1 5 10 15Leu Ala Ala Leu Leu Leu Ala Ala Arg Gly Ala Ala Ala
Gln Glu Thr 20 25 30Glu Leu Ser Val Ser Ala Glu Leu Val Pro Thr Ser
Ser Trp Asn Ile 35 40 45Ser Ser Glu Leu Asn Lys Asp Ser Tyr Leu Thr
Leu Asp Glu Pro Met 50 55 60Asn Asn Ile Thr Thr Ser Leu Gly Gln Thr
Ala Glu Leu His Cys Lys65 70 75 80Val
Ser Gly Asn Pro Pro Pro Thr Ile Arg Trp Phe Lys Asn Asp Ala 85 90
95Pro Val Val Gln Glu Pro Arg Arg Leu Ser Phe Arg Ser Thr Ile Tyr
100 105 110Gly Ser Arg Leu Arg Ile Arg Asn Leu Asp Thr Thr Asp Thr
Gly Tyr 115 120 125Phe Gln Cys Val Ala Thr Asn Gly Lys Glu Val Val
Ser Ser Thr Gly 130 135 140Val Leu Phe Val Lys Phe Gly Pro Pro Pro
Thr Ala Ser Pro Gly Tyr145 150 155 160Ser Asp Glu Tyr Glu Glu Asp
Gly Phe Cys Gln Pro Tyr Arg Gly Ile 165 170 175Ala Cys Ala Arg Phe
Ile Gly Asn Arg Thr Val Tyr Met Glu Ser Leu 180 185 190His Met Gln
Gly Glu Ile Glu Asn Gln Ile Thr Ala Ala Phe Thr Met 195 200 205Ile
Gly Thr Ser Ser His Leu Ser Asp Lys Cys Ser Gln Phe Ala Ile 210 215
220Pro Ser Leu Cys His Tyr Ala Phe Pro Tyr Cys Asp Glu Thr Ser
Ser225 230 235 240Val Pro Lys Pro Arg Asp Leu Cys Arg Asp Glu Cys
Glu Ile Leu Glu 245 250 255Asn Val Leu Cys Gln Thr Glu Tyr Ile Phe
Ala Arg Ser Asn Pro Met 260 265 270Ile Leu Met Arg Leu Lys Leu Pro
Asn Cys Glu Asp Leu Pro Gln Pro 275 280 285Glu Ser Pro Glu Ala Ala
Asn Cys Ile Arg Ile Gly Ile Pro Met Ala 290 295 300Asp Pro Ile Asn
Lys Asn His Lys Cys Tyr Asn Ser Thr Gly Val Asp305 310 315 320Tyr
Arg Gly Thr Val Ser Val Thr Lys Ser Gly Arg Gln Cys Gln Pro 325 330
335Trp Asn Ser Gln Tyr Pro His Thr His Thr Phe Thr Ala Leu Arg Phe
340 345 350Pro Glu Leu Asn Gly Gly His Ser Tyr Cys Arg Asn Pro Gly
Asn Gln 355 360 365Lys Glu Ala Pro Trp Cys Phe Thr Leu Asp Glu Asn
Phe Lys Ser Asp 370 375 380Leu Cys Asp Ile Pro Ala Cys Asp Ser Lys
Asp Ser Lys Glu Lys Asn385 390 395 400Lys Met Glu Ile Leu Tyr Ile
Leu Val Pro Ser Val Ala Ile Pro Leu 405 410 415Ala Ile Ala Leu Leu
Phe Phe Phe Ile Cys Val Cys Arg Asn Asn Gln 420 425 430Lys Ser Ser
Ser Pro Pro Val Gln Arg Gln Pro Lys His Val Arg Gly 435 440 445Gln
Asn Val Glu Met Ser Met Leu Asn Ala Tyr Lys Pro Lys Ser Lys 450 455
460Ala Lys Glu Leu Pro Leu Ser Ala Val Arg Phe Met Glu Glu Leu
Gly465 470 475 480Glu Cys Ala Phe Gly Lys Ile Tyr Lys Gly His Leu
Tyr Leu Pro Gly 485 490 495Met Asp His Ala Gln Leu Val Ala Ile Lys
Thr Leu Lys Asp Tyr Asn 500 505 510Asn Pro Gln Gln Trp Thr Glu Phe
Gln Gln Glu Ala Ser Leu Met Ala 515 520 525Glu Leu His His Pro Asn
Ile Val Cys Leu Leu Gly Ala Val Thr Gln 530 535 540Glu Gln Pro Val
Cys Met Leu Phe Glu Tyr Met Asn Gln Gly Asp Leu545 550 555 560His
Glu Phe Leu Ile Met Arg Ser Pro His Ser Asp Val Gly Cys Ser 565 570
575Ser Asp Glu Asp Gly Thr Val Lys Ser Ser Leu Asp His Gly Asp Phe
580 585 590Leu His Ile Ala Ile Gln Ile Ala Ala Gly Met Glu Tyr Leu
Ser Ser 595 600 605His Phe Phe Val His Lys Asp Leu Ala Ala Arg Asn
Ile Leu Ile Gly 610 615 620Glu Gln Leu His Val Lys Ile Ser Asp Leu
Gly Leu Ser Arg Glu Ile625 630 635 640Tyr Ser Ala Asp Tyr Tyr Arg
Val Gln Ser Lys Ser Leu Leu Pro Ile 645 650 655Arg Trp Met Pro Pro
Glu Ala Ile Met Tyr Gly Lys Phe Ser Ser Asp 660 665 670Ser Asp Ile
Trp Ser Phe Gly Val Val Leu Trp Glu Ile Phe Ser Phe 675 680 685Gly
Leu Gln Pro Tyr Tyr Gly Phe Ser Asn Gln Glu Val Ile Glu Met 690 695
700Val Arg Lys Arg Gln Leu Leu Pro Cys Ser Glu Asp Cys Pro Pro
Arg705 710 715 720Met Tyr Ser Leu Met Thr Glu Cys Trp Asn Glu Ile
Pro Ser Arg Arg 725 730 735Pro Arg Phe Lys Asp Ile His Val Arg Leu
Arg Ser Trp Glu Gly Leu 740 745 750Ser Ser His Thr Ser Ser Thr Thr
Pro Ser Gly Gly Asn Ala Thr Thr 755 760 765Gln Thr Thr Ser Leu Ser
Ala Ser Pro Val Ser Asn Leu Ser Asn Pro 770 775 780Arg Tyr Pro Asn
Tyr Ile Phe Pro Ser Gln Gly Ile Thr Pro Gln Gly785 790 795 800Gln
Ile Ala Gly Phe Ile Gly Pro Pro Ile Pro Gln Asn Gln Arg Phe 805 810
815Ile Pro Ile Asn Gly Tyr Pro Ile Pro Pro Gly Tyr Ala Ala Phe Pro
820 825 830Ala Ala His Tyr Gln Pro Thr Gly Pro Pro Arg Val Ile Gln
His Cys 835 840 845Pro Pro Pro Lys Ser Arg Ser Pro Ser Ser Ala Ser
Gly Ser Thr Ser 850 855 860Thr Gly His Val Thr Ser Leu Pro Ser Ser
Gly Ser Asn Gln Glu Ala865 870 875 880Asn Ile Pro Leu Leu Pro His
Met Ser Ile Pro Asn His Pro Gly Gly 885 890 895Met Gly Ile Thr Val
Phe Gly Asn Lys Ser Gln Lys Pro Tyr Lys Ile 900 905 910Asp Ala Lys
Gln Ala Ser Leu Leu Gly Asp Ala Asn Ile His Gly His 915 920 925Thr
Glu Ser Met Ile Ser Ala Glu Leu 930 935217923PRTmacaca
fasicularisMISC_FEATUREROR1 (Uniprot A0A2K5WTX7) 217Met Leu Arg Thr
Ala His Lys Leu Leu Tyr Leu Ile Leu Pro Leu Ser1 5 10 15Phe Ser Leu
Pro Phe Phe Phe Phe Ser Glu Thr Glu Leu Ser Val Ser 20 25 30Ala Glu
Leu Val Pro Thr Ser Ser Trp Asn Ile Ser Ser Glu Leu Asn 35 40 45Lys
Asp Ser Tyr Leu Thr Leu Asp Glu Pro Met Asn Asn Ile Thr Thr 50 55
60Ser Leu Gly Gln Thr Ala Glu Leu His Cys Lys Val Ser Gly Asn Pro65
70 75 80Pro Pro Thr Ile Arg Trp Phe Lys Asn Asp Ala Pro Val Val Gln
Glu 85 90 95Pro Arg Arg Leu Ser Phe Arg Ser Thr Ile Tyr Gly Ser Arg
Leu Arg 100 105 110Ile Arg Asn Leu Asp Thr Thr Asp Thr Gly Tyr Phe
Gln Cys Val Ala 115 120 125Thr Asn Gly Lys Glu Val Val Ser Ser Thr
Gly Val Leu Phe Val Lys 130 135 140Phe Gly Lys Asp Glu Tyr Glu Glu
Asp Gly Phe Cys Gln Pro Tyr Arg145 150 155 160Gly Ile Ala Cys Ala
Arg Phe Ile Gly Asn Arg Thr Val Tyr Met Glu 165 170 175Ser Leu His
Met Gln Gly Glu Ile Glu Asn Gln Ile Thr Ala Ala Phe 180 185 190Thr
Met Ile Gly Thr Ser Ser His Leu Ser Asp Lys Cys Ser Gln Phe 195 200
205Ala Ile Pro Ser Leu Cys His Tyr Ala Phe Pro Tyr Cys Asp Glu Thr
210 215 220Ser Ser Val Pro Lys Pro Arg Asp Leu Cys Arg Asp Glu Cys
Glu Ile225 230 235 240Leu Glu Asn Val Leu Cys Gln Thr Glu Tyr Ile
Phe Ala Arg Ser Asn 245 250 255Pro Met Ile Leu Met Arg Leu Lys Leu
Pro Asn Cys Glu Asp Leu Pro 260 265 270Gln Pro Glu Ser Pro Glu Ala
Ala Asn Cys Ile Arg Ile Gly Ile Pro 275 280 285Met Ala Asp Pro Ile
Asn Lys Asn His Lys Cys Tyr Asn Ser Thr Gly 290 295 300Val Asp Tyr
Arg Gly Thr Val Ser Val Thr Lys Ser Gly Arg Gln Cys305 310 315
320Gln Pro Trp Asn Ser Gln Tyr Pro His Thr His Thr Phe Thr Ala Leu
325 330 335Arg Phe Pro Glu Leu Asn Gly Gly His Ser Tyr Cys Arg Asn
Pro Gly 340 345 350Asn Gln Lys Glu Ala Pro Trp Cys Phe Thr Leu Asp
Glu Asn Phe Lys 355 360 365Ser Asp Leu Cys Asp Ile Pro Ala Cys Asp
Ser Lys Asp Ser Lys Glu 370 375 380Lys Asn Lys Met Glu Ile Leu Tyr
Ile Leu Val Pro Ser Val Ala Ile385 390 395 400Pro Leu Ala Ile Ala
Leu Leu Phe Phe Phe Ile Cys Val Cys Arg Asn 405 410 415Asn Gln Lys
Ser Ser Ser Pro Pro Val Gln Arg Gln Pro Lys His Val 420 425 430Arg
Gly Gln Asn Val Glu Met Ser Met Leu Asn Ala Tyr Lys Pro Lys 435 440
445Ser Lys Ala Lys Glu Leu Pro Leu Ser Ala Val Arg Phe Met Glu Glu
450 455 460Leu Gly Glu Cys Ala Phe Gly Lys Ile Tyr Lys Gly His Leu
Tyr Leu465 470 475 480Pro Gly Met Asp His Ala Gln Leu Val Ala Ile
Lys Thr Leu Lys Asp 485 490 495Tyr Asn Asn Pro Gln Gln Trp Thr Glu
Phe Gln Gln Glu Ala Ser Leu 500 505 510Met Ala Glu Leu His His Pro
Asn Ile Val Cys Leu Leu Gly Ala Val 515 520 525Thr Gln Glu Gln Pro
Val Cys Met Leu Phe Glu Tyr Met Asn Gln Gly 530 535 540Asp Leu His
Glu Phe Leu Ile Met Arg Ser Pro His Ser Asp Val Gly545 550 555
560Cys Ser Ser Asp Glu Asp Gly Thr Val Lys Ser Ser Leu Asp His Gly
565 570 575Asp Phe Leu His Ile Ala Ile Gln Ile Ala Ala Gly Met Glu
Tyr Leu 580 585 590Ser Ser His Phe Phe Val His Lys Asp Leu Ala Ala
Arg Asn Ile Leu 595 600 605Ile Gly Glu Gln Leu His Val Lys Ile Ser
Asp Leu Gly Leu Ser Arg 610 615 620Glu Ile Tyr Ser Ala Asp Tyr Tyr
Arg Val Gln Ser Lys Ser Leu Leu625 630 635 640Pro Ile Arg Trp Met
Pro Pro Glu Ala Ile Met Tyr Gly Lys Phe Ser 645 650 655Ser Asp Ser
Asp Ile Trp Ser Phe Gly Val Val Leu Trp Glu Ile Phe 660 665 670Ser
Phe Gly Leu Gln Pro Tyr Tyr Gly Phe Ser Asn Gln Glu Val Ile 675 680
685Glu Met Val Arg Lys Arg Gln Leu Leu Pro Cys Ser Glu Asp Cys Pro
690 695 700Pro Arg Met Tyr Ser Leu Met Thr Glu Cys Trp Asn Glu Ile
Pro Ser705 710 715 720Arg Arg Pro Arg Phe Lys Asp Ile His Val Arg
Leu Arg Ser Trp Glu 725 730 735Gly Leu Ser Ser His Thr Ser Ser Thr
Thr Pro Ser Gly Gly Asn Ala 740 745 750Thr Thr Gln Thr Thr Ser Leu
Ser Ala Ser Pro Val Ser Asn Leu Ser 755 760 765Asn Pro Arg Tyr Pro
Asn Tyr Ile Phe Pro Ser Gln Gly Ile Thr Pro 770 775 780Gln Gly Gln
Ile Ala Gly Phe Ile Gly Pro Pro Ile Pro Gln Asn Gln785 790 795
800Arg Phe Ile Pro Ile Asn Gly Tyr Pro Ile Pro Pro Gly Tyr Ala Ala
805 810 815Phe Pro Ala Ala His Tyr Gln Pro Thr Gly Pro Pro Arg Val
Ile Gln 820 825 830His Cys Pro Pro Pro Lys Ser Arg Ser Pro Ser Ser
Ala Ser Gly Ser 835 840 845Thr Ser Thr Gly His Val Thr Ser Leu Pro
Ser Ser Gly Ser Asn Gln 850 855 860Glu Ala Asn Ile Pro Leu Leu Pro
His Met Ser Ile Pro Asn His Pro865 870 875 880Gly Gly Met Gly Ile
Thr Val Phe Gly Asn Lys Ser Gln Lys Pro Tyr 885 890 895Lys Ile Asp
Ala Lys Gln Ala Ser Leu Leu Gly Asp Ala Asn Ile His 900 905 910Gly
His Thr Glu Ser Met Ile Ser Ala Glu Leu 915 920218882PRTmacaca
fasicularisMISC_FEATUREROR1 (Uniprot A0A2K5WTX4) 218Ser Tyr Leu Thr
Leu Asp Glu Pro Met Asn Asn Ile Thr Thr Ser Leu1 5 10 15Gly Gln Thr
Ala Glu Leu His Cys Lys Val Ser Gly Asn Pro Pro Pro 20 25 30Thr Ile
Arg Trp Phe Lys Asn Asp Ala Pro Val Val Gln Glu Pro Arg 35 40 45Arg
Leu Ser Phe Arg Ser Thr Ile Tyr Gly Ser Arg Leu Arg Ile Arg 50 55
60Asn Leu Asp Thr Thr Asp Thr Gly Tyr Phe Gln Cys Val Ala Thr Asn65
70 75 80Gly Lys Glu Val Val Ser Ser Thr Gly Val Leu Phe Val Lys Phe
Gly 85 90 95Pro Pro Pro Thr Ala Ser Pro Gly Tyr Ser Asp Glu Tyr Glu
Glu Asp 100 105 110Gly Phe Cys Gln Pro Tyr Arg Gly Ile Ala Cys Ala
Arg Phe Ile Gly 115 120 125Asn Arg Thr Val Tyr Met Glu Ser Leu His
Met Gln Gly Glu Ile Glu 130 135 140Asn Gln Ile Thr Ala Ala Phe Thr
Met Ile Gly Thr Ser Ser His Leu145 150 155 160Ser Asp Lys Cys Ser
Gln Phe Ala Ile Pro Ser Leu Cys His Tyr Ala 165 170 175Phe Pro Tyr
Cys Asp Glu Thr Ser Ser Val Pro Lys Pro Arg Asp Leu 180 185 190Cys
Arg Asp Glu Cys Glu Ile Leu Glu Asn Val Leu Cys Gln Thr Glu 195 200
205Tyr Ile Phe Ala Arg Ser Asn Pro Met Ile Leu Met Arg Leu Lys Leu
210 215 220Pro Asn Cys Glu Asp Leu Pro Gln Pro Glu Ser Pro Glu Ala
Ala Asn225 230 235 240Cys Ile Arg Ile Gly Ile Pro Met Ala Asp Pro
Ile Asn Lys Asn His 245 250 255Lys Cys Tyr Asn Ser Thr Gly Val Asp
Tyr Arg Gly Thr Val Ser Val 260 265 270Thr Lys Ser Gly Arg Gln Cys
Gln Pro Trp Asn Ser Gln Tyr Pro His 275 280 285Thr His Thr Phe Thr
Ala Leu Arg Phe Pro Glu Leu Asn Gly Gly His 290 295 300Ser Tyr Cys
Arg Asn Pro Gly Asn Gln Lys Glu Ala Pro Trp Cys Phe305 310 315
320Thr Leu Asp Glu Asn Phe Lys Ser Asp Leu Cys Asp Ile Pro Ala Cys
325 330 335Asp Ser Lys Asp Ser Lys Glu Lys Asn Lys Met Glu Ile Leu
Tyr Ile 340 345 350Leu Val Pro Ser Val Ala Ile Pro Leu Ala Ile Ala
Leu Leu Phe Phe 355 360 365Phe Ile Cys Val Cys Arg Asn Asn Gln Lys
Ser Ser Ser Pro Pro Val 370 375 380Gln Arg Gln Pro Lys His Val Arg
Gly Gln Asn Val Glu Met Ser Met385 390 395 400Leu Asn Ala Tyr Lys
Pro Lys Ser Lys Ala Lys Glu Leu Pro Leu Ser 405 410 415Ala Val Arg
Phe Met Glu Glu Leu Gly Glu Cys Ala Phe Gly Lys Ile 420 425 430Tyr
Lys Gly His Leu Tyr Leu Pro Gly Met Asp His Ala Gln Leu Val 435 440
445Ala Ile Lys Thr Leu Lys Asp Tyr Asn Asn Pro Gln Gln Trp Thr Glu
450 455 460Phe Gln Gln Glu Ala Ser Leu Met Ala Glu Leu His His Pro
Asn Ile465 470 475 480Val Cys Leu Leu Gly Ala Val Thr Gln Glu Gln
Pro Val Cys Met Leu 485 490 495Phe Glu Tyr Met Asn Gln Gly Asp Leu
His Glu Phe Leu Ile Met Arg 500 505 510Ser Pro His Ser Asp Val Gly
Cys Ser Ser Asp Glu Asp Gly Thr Val 515 520 525Lys Ser Ser Leu Asp
His Gly Asp Phe Leu His Ile Ala Ile Gln Ile 530 535 540Ala Ala Gly
Met Glu Tyr Leu Ser Ser His Phe Phe Val His Lys Asp545 550 555
560Leu Ala Ala Arg Asn Ile Leu Ile Gly Glu Gln Leu His Val Lys Ile
565 570 575Ser Asp Leu Gly Leu Ser Arg Glu Ile Tyr Ser Ala Asp Tyr
Tyr Arg 580 585 590Val Gln Ser Lys Ser Leu Leu Pro Ile Arg Trp Met
Pro Pro Glu Ala 595 600 605Ile Met Tyr Gly Lys Phe Ser Ser Asp Ser
Asp Ile Trp Ser Phe Gly 610 615 620Val Val Leu Trp Glu Ile Phe Ser
Phe Gly Leu Gln Pro Tyr Tyr Gly625 630 635 640Phe Ser Asn Gln Glu
Val Ile Glu Met Val Arg Lys Arg Gln Leu Leu 645 650 655Pro Cys Ser
Glu Asp Cys Pro Pro Arg Met Tyr Ser Leu Met Thr Glu 660 665 670Cys
Trp Asn Glu Ile Pro Ser Arg Arg Pro
Arg Phe Lys Asp Ile His 675 680 685Val Arg Leu Arg Ser Trp Glu Gly
Leu Ser Ser His Thr Ser Ser Thr 690 695 700Thr Pro Ser Gly Gly Asn
Ala Thr Thr Gln Thr Thr Ser Leu Ser Ala705 710 715 720Ser Pro Val
Ser Asn Leu Ser Asn Pro Arg Tyr Pro Asn Tyr Ile Phe 725 730 735Pro
Ser Gln Gly Ile Thr Pro Gln Gly Gln Ile Ala Gly Phe Ile Gly 740 745
750Pro Pro Ile Pro Gln Asn Gln Arg Phe Ile Pro Ile Asn Gly Tyr Pro
755 760 765Ile Pro Pro Gly Tyr Ala Ala Phe Pro Ala Ala His Tyr Gln
Pro Thr 770 775 780Gly Pro Pro Arg Val Ile Gln His Cys Pro Pro Pro
Lys Ser Arg Ser785 790 795 800Pro Ser Ser Ala Ser Gly Ser Thr Ser
Thr Gly His Val Thr Ser Leu 805 810 815Pro Ser Ser Gly Ser Asn Gln
Glu Ala Asn Ile Pro Leu Leu Pro His 820 825 830Met Ser Ile Pro Asn
His Pro Gly Gly Met Gly Ile Thr Val Phe Gly 835 840 845Asn Lys Ser
Gln Lys Pro Tyr Lys Ile Asp Ala Lys Gln Ala Ser Leu 850 855 860Leu
Gly Asp Ala Asn Ile His Gly His Thr Glu Ser Met Ile Ser Ala865 870
875 880Glu Leu219937PRTmus musculusMISC_FEATUREROR1 (Uniprot
Q9Z139) 219Met His Arg Pro Arg Arg Arg Gly Thr Arg Pro Pro Pro Leu
Ala Leu1 5 10 15Leu Ala Ala Leu Leu Leu Ala Ala Arg Gly Ala Asp Ala
Gln Glu Thr 20 25 30Glu Leu Ser Val Ser Ala Glu Leu Val Pro Thr Ser
Ser Trp Asn Thr 35 40 45Ser Ser Glu Ile Asp Lys Gly Ser Tyr Leu Thr
Leu Asp Glu Pro Met 50 55 60Asn Asn Ile Thr Thr Ser Leu Gly Gln Thr
Ala Glu Leu His Cys Lys65 70 75 80Val Ser Gly Asn Pro Pro Pro Ser
Ile Arg Trp Phe Lys Asn Asp Ala 85 90 95Pro Val Val Gln Glu Pro Arg
Arg Ile Ser Phe Arg Ala Thr Asn Tyr 100 105 110Gly Ser Arg Leu Arg
Ile Arg Asn Leu Asp Thr Thr Asp Thr Gly Tyr 115 120 125Phe Gln Cys
Val Ala Thr Asn Gly Lys Lys Val Val Ser Thr Thr Gly 130 135 140Val
Leu Phe Val Lys Phe Gly Pro Pro Pro Thr Ala Ser Pro Gly Ser145 150
155 160Ser Asp Glu Tyr Glu Glu Asp Gly Phe Cys Gln Pro Tyr Arg Gly
Ile 165 170 175Ala Cys Ala Arg Phe Ile Gly Asn Arg Thr Val Tyr Met
Glu Ser Leu 180 185 190His Met Gln Gly Glu Ile Glu Asn Gln Ile Thr
Ala Ala Phe Thr Met 195 200 205Ile Gly Thr Ser Ser His Leu Ser Asp
Lys Cys Ser Gln Phe Ala Ile 210 215 220Pro Ser Leu Cys His Tyr Ala
Phe Pro Tyr Cys Asp Glu Thr Ser Ser225 230 235 240Val Pro Lys Pro
Arg Asp Leu Cys Arg Asp Glu Cys Glu Val Leu Glu 245 250 255Asn Val
Leu Cys Gln Thr Glu Tyr Ile Phe Ala Arg Ser Asn Pro Met 260 265
270Ile Leu Met Arg Leu Lys Leu Pro Asn Cys Glu Asp Leu Pro Gln Pro
275 280 285Glu Ser Pro Glu Ala Ala Asn Cys Ile Arg Ile Gly Ile Pro
Met Ala 290 295 300Asp Pro Ile Asn Lys Asn His Lys Cys Tyr Asn Ser
Thr Gly Val Asp305 310 315 320Tyr Arg Gly Thr Val Ser Val Thr Lys
Ser Gly Arg Gln Cys Gln Pro 325 330 335Trp Asn Ser Gln Tyr Pro His
Thr His Ser Phe Thr Ala Leu Arg Phe 340 345 350Pro Glu Leu Asn Gly
Gly His Ser Tyr Cys Arg Asn Pro Gly Asn Gln 355 360 365Lys Glu Ala
Pro Trp Cys Phe Thr Leu Asp Glu Asn Phe Lys Ser Asp 370 375 380Leu
Cys Asp Ile Pro Ala Cys Asp Ser Lys Asp Ser Lys Glu Lys Asn385 390
395 400Lys Met Glu Ile Leu Tyr Ile Leu Val Pro Ser Val Ala Ile Pro
Leu 405 410 415Ala Ile Ala Phe Leu Phe Phe Phe Ile Cys Val Cys Arg
Asn Asn Gln 420 425 430Lys Ser Ser Ser Pro Pro Val Gln Arg Gln Pro
Lys Pro Val Arg Gly 435 440 445Gln Asn Val Glu Met Ser Met Leu Asn
Ala Tyr Lys Pro Lys Ser Lys 450 455 460Ala Lys Glu Leu Pro Leu Ser
Ala Val Arg Phe Met Glu Glu Leu Gly465 470 475 480Glu Cys Thr Phe
Gly Lys Ile Tyr Lys Gly His Leu Tyr Leu Pro Gly 485 490 495Met Asp
His Ala Gln Leu Val Ala Ile Lys Thr Leu Lys Asp Tyr Asn 500 505
510Asn Pro Gln Gln Trp Thr Glu Phe Gln Gln Glu Ala Ser Leu Met Ala
515 520 525Glu Leu His His Pro Asn Ile Val Cys Leu Leu Gly Ala Val
Thr Gln 530 535 540Glu Gln Pro Val Cys Met Leu Phe Glu Tyr Met Asn
Gln Gly Asp Leu545 550 555 560His Glu Phe Leu Ile Met Arg Ser Pro
His Ser Asp Val Gly Cys Ser 565 570 575Ser Asp Glu Asp Gly Thr Val
Lys Ser Ser Leu Asp His Gly Asp Phe 580 585 590Leu His Ile Ala Ile
Gln Ile Ala Ala Gly Met Glu Tyr Leu Ser Ser 595 600 605His Phe Phe
Val His Lys Asp Leu Ala Ala Arg Asn Ile Leu Ile Gly 610 615 620Glu
Gln Leu His Val Lys Ile Ser Asp Leu Gly Leu Ser Arg Glu Ile625 630
635 640Tyr Ser Ala Asp Tyr Tyr Arg Val Gln Ser Lys Ser Ser Leu Pro
Ile 645 650 655Arg Trp Met Pro Pro Glu Ala Ile Met Tyr Gly Lys Phe
Ser Ser Asp 660 665 670Ser Asp Ile Trp Ser Phe Gly Val Val Leu Trp
Glu Ile Phe Ser Phe 675 680 685Gly Leu Gln Pro Tyr Tyr Gly Phe Ser
Asn Gln Glu Val Ile Glu Met 690 695 700Val Arg Lys Arg Gln Leu Leu
Pro Cys Ser Glu Asp Cys Pro Pro Arg705 710 715 720Met Tyr Ser Leu
Met Thr Glu Cys Trp Asn Glu Ile Pro Ser Arg Arg 725 730 735Pro Arg
Phe Lys Asp Ile His Val Arg Leu Arg Ser Trp Glu Gly Leu 740 745
750Ser Ser His Thr Ser Ser Thr Thr Pro Ser Gly Gly Asn Ala Thr Thr
755 760 765Gln Thr Thr Ser Leu Ser Ala Ser Pro Val Ser Asn Leu Ser
Asn Pro 770 775 780Arg Phe Pro Asn Tyr Met Phe Pro Ser Gln Gly Ile
Thr Pro Gln Gly785 790 795 800Gln Ile Ala Gly Phe Ile Gly Pro Ala
Ile Pro Gln Asn Gln Arg Phe 805 810 815Ile Pro Ile Asn Gly Tyr Pro
Ile Pro Pro Gly Tyr Ala Ala Phe Pro 820 825 830Ala Ala His Tyr Gln
Pro Ala Gly Pro Pro Arg Val Ile Gln His Cys 835 840 845Pro Pro Pro
Lys Ser Arg Ser Pro Ser Ser Ala Ser Gly Ser Thr Ser 850 855 860Thr
Gly His Val Ala Ser Leu Pro Ser Ser Gly Ser Asn Gln Glu Ala865 870
875 880Asn Val Pro Leu Leu Pro His Met Ser Ile Pro Asn His Pro Gly
Gly 885 890 895Met Gly Ile Thr Val Phe Gly Asn Lys Ser Gln Lys Pro
Tyr Lys Ile 900 905 910Asp Ser Lys Gln Ser Ser Leu Leu Gly Asp Ser
His Ile His Gly His 915 920 925Thr Glu Ser Met Ile Ser Ala Glu Val
930 935
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